Insulin-like growth factor binding protein (IGFBF-5)

ABSTRACT

A purified insulin-like growth factor binding protein (IGFBP) selected from the group consisting of insulin-like growth factor binding protein having an amino acid sequence that, preferably, is at least 70% homologous to the amino acid sequence of FIG.  1  and fragments thereof that are capable of binding to an antibody specific for the protein or to an insulin-like growth factor is described. This new IGFBP is designated herein as IGFBP-5. Recombinant DNA molecules encoding the binding proteins and subsequences thereof are also described along, with recombinant microorganisms and cell lines containing the DNA molecules and methods for producing the binding proteins using recombinant hosts containing the relevant DNA molecules. Antibodies to the protein, useful in various diagnostic applications, are also described.

This application is a continuation, of application Ser. No. 07/638,628,filed Jan. 8, 1991 now abandoned This application is a continuation ofapplication Ser. No. 08/142,848, filed Oct. 26, 1993 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Disclosure

The present invention relates generally to production of polypeptidesfrom recombinant DNA molecules encoding such polypeptides. Morespecifically, this invention relates to a new insulin-like growth factorbinding protein (designated herein as IGFBP-5), recombinant DNAmolecules encoding this polypeptide, and methods for producing IGFBP-5from recombinant host cells.

2. Description of the Related Art

Insulin-like growth factors (IGFS) are low molecular weight polypeptidehormones with structural homology to proinsulin. Two different IGFs areknown, namely IGF-I and IGF-II, which are mitogenic in vitro for a widevariety of cells in tissue culture. Both IGFs stimulate in vitro thegrowth of various tissues and in particular they induce collagensynthesis. IGF-I mediates the growth promoting effect of growth hormonein chondrogenesis and bone formation and is therefore essential fornormal growth of an individual. This is demonstrated by the fact thatpygmies and toy poodles are deficient in IGF-I but have normal growthhormone level in their serum. IGF-II is believed to play a key role infetal development and nerve growth.

In addition to their primary effect on skeletal tissue IGFs also exhibitgrowth-stimulating functions on other tissues. Wound fibroblasts areknown to produce IGFs which are effective in stimulating fibroblasts togrow and synthesize collagen, a structural protein normally required forwound healing. Vascularization of the wound tissue is also induced.Further, it has also been found that IGFs have an erythropoietin-likeactivity in that they induce hematopoiesis.

Recent studies have also demonstrated that IGFs produced by certaincancer cells, e.g. breast and kidney cancer cells, auto-stimulate theproliferation of cancer cells and the vascular and fibrous tissuesrequired to support the growth of cancer tissues.

In addition to this, both IGFs show a spectrum of metabolic activitiessimilar to those of insulin, in that they stimulate, in particular, thetransport and metabolism of glucose. The biological effects of IGFs andinsulin are mediated through their binding to specific receptors. Inparticular, both IGFs have the ability to bind to the insulin receptorwith approximately 100-fold lower affinity than does insulin.

Both IGFs have a concentration in blood approximately a hundred-foldhigher than that of insulin. Hypoglycemia is prevented by a regulatorymechanism which involves carrier proteins present in blood and able toform complexes with IGFs. Thus, IGFs circulate in the blood in the formof a complex which has no insulin-like activity.

Through their association with carrier proteins (hereinafter referred toas IGF binding proteins or ICFBPs), binding of IGFs to cell surfacereceptors is inhibited. It has also been demonstrated that anotherfunction of the IGF binding proteins is to increase the short half-lifeof IGFs, which are subjected to rapid proteolytic degradation whenpresent in the free form in blood.

In accordance with the foregoing, IGFs may be useful in vitro tostimulate a) the growth of animals and humans with growth hormonedeficiency, b) tissue regeneration, such as erythropoiesis andchondrogenesis, c) wound healing and d) the functions of various organse.g. liver or kidney. As a result of their chondrogenesis stimulatingactivity, IGFs are of particularly suitable use for bone formation, e.g.in the treatment of osteoporosis.

IGFs for use in the above-referred treatments are advantageouslyadministered to a subject in association with at least one IGF bindingprotein. Through their association with carrier proteins (hereinafterreferred to as IGF binding proteins or IGFBPs), binding of IGFs to cellsurface receptors is inhibited. It has also been demonstrated thatanother function of the IGF binding proteins is to increase the shorthalf-life of IGFs, which are subjected to rapid proteolytic degradationwhen present in the free form in blood.

Administration of the combination of IGF and an IGF binding protein,rather than IGF alone, has beneficial effects including the preventionof hypoglycemia and possible mitogenic effects at injection sites andthe prolongation of IGF half-life. Furthermore, it has been found thatbinding proteins are also useful for potentiating the erythropoietinlike-effect of IGF-I. The binding proteins may also be useful fortargeting IGFs to specific tissues.

When administered alone, i.e., without any IGF, the binding proteins mayalso be therapeutically useful for blocking the adverse effects of IGFs,such as those which occur when IGFs are produced in excess, e.g. freeIGFs secreted by certain cancer cells e.g. hormone-producing cancercells such as breast or kidney cancer cells. IGF binding protein therapymay also prevent blindness as a secondary effect of diabeticproliferation retinopathy. Indeed it has been shown that IGFs may be oneof the factors stimulating endothelial and fibroblast proliferation indiabetic retinopathy.

Another therapeutic use of IGFBPs is the control of excessive growth inIGF binding protein-deficient subjects, since it is very likely thathigh IGF levels combined with abnormally low levels of binding proteinare responsible for excessive growth.

Known forms of IGFBPs include IGFBP-1, having a molecular weight ofapproximately 30-40 kd in humans. See, e.g., Povoa, G. et al., Eur. J.Biochem (1984) 144:199-204, relates to IGFBP-1, isolated and purifiedfrom amniotic fluid; Koistinen, R. et al., Endocrinology (1986)118:1375-378, relates to IGFBP-1 isolated and purified from: humanplacenta; Powell, D. R. et al., J. Chromatogr. (1987) 420:163-170,relates to a 30-40 kd IGFBP-1 isolated and purified from conditionedmedium of hepatoma G2 (Hep-G2) cells; Lee, Y. L. et al., Mol.Endocrinol. (1988) 2:404-411, relates to an amino acid sequence ofIGFBP-1 isolated from Hep-G2 cells; Brinkman, A. et al., The EMBOJournal (1988) 7: 2417-2423, relates to an IGFBP-1 placental cDNAlibrary; Brewer, M. T. et al., Bioch. Biophys. Res. Com. (1988)152:1289-1297, pertains to nucleotide and amino acid sequences forIGFBP-1 cloned from a human uterine decidua library; WO89/09792,published Oct. 19, 1990, Clemmons, D. R., et al., pertains to cDNAsequences and cloning vectors for IGFBP-1 and IGFBP-2; WO89/08667,published Sept. 21, 1989, Drop, L. S., et al., relates to an amino acidsequence of insulin-like-growth factor binding protein 1 (IGFBP-1);WO89/09268, published Oct. 5, 1989, Baxter, R. C., relates to a cDNAsequence of IGFBP-1 and methods of expression for IGFBP-1.

IGFBP-2 has a molecular weight of approximately 33-36 kd. See, e.g.,Binkert, C. et al., The EMBO Journal (1989) 8:2497-2502, relates to anucleotide and deduced amino acid sequence for IGFBP-2.

IGFBP-3 has a molecular weight of 150 kd. See, e.g., Baxter, R. C. etal., Bioch. Biopys. Res. Com. (1986) 139:1256-1261, pertains to a 53 kdsubunit of IGFBP-3 that was purified from human serum; Wood, W. I. etal., Mol. Endocrinol. (1988) 2:1176-1185, relates to a full length aminoacid sequence for IGFBP-3 and cellular expression of the cloned IGFBP-3cDNA in mammalian tissue culture cells; WO90/00569, published Jan. 25,1990, Baxter, R. C., relates to isolating from human plasma anacid-labile subunit (ALS) of (IGFBP) complex and, the particular aminoacid sequence for ALS pertains to a subunit of IGFBP-3.

For nonhuman forms, see, e.g., Mottola, C. et al., Journ. of Biol. Chem.(1986) 261: 11180-11188, relates to a non-human form of IGFBP that wasisolated in conditioned medium from rat liver BRL-3A cells and has amolecular weight of approximately 33-36 kd; Lyons, R. M. et al., Mol.Cell. Endocrinol. (1986) 45: 263-270, relates to a 34 kd cloned BRL-3Arat liver cell protein designated MCP; EPO Publ. No. 369 943, publishedMay 23 1990, Binkert, C., et al., relates to a cDNA sequence of the ratBRL-3A binding protein and uses this sequence to screen three human cDNAlibraries.

Mohan, S. et al., Proc. Natl. Acad. Scii. (1989) 86:8338-8342, relatesto an N-terminal amino acid sequence for an IGFBP (designated therein asIGFBP-4 but, using Applicants' terminology as defined in theapplications listed below, actually corresponding to IGFBP-5) isolatedfrom medium conditioned by human osteosarcoma cells and Shimasaki, S. etal., Mol. Endocrinology (1990) 4:1451-1458, pertains to IGFBP cDNAsencoding an IGFBP (designated therein as IGFBP-4 but, using Applicants'terminology, actually corresponding to IGFBP-5) from rat and human.

Copending application Ser. No. 07/574,613, filed Aug. 28, 1990, which isco-owned by the present assignee, relates to IGFBP-6 and IGFBP-4 geneticmaterial and amino acid sequences; copending application Ser. No.07/576,648, filed Aug. 31, 1990, which is co-owned by the presentassignee, relates to IGFBP-6 amino acid sequences; copending applicationSer. No. 07/576,629, filed Aug. 31, 1990, which is co-owned by thepresent assignee, relates to genetic material encoding IGFBP-6;copending application Ser. No. 07/577,391, filed Aug. 31, 1990, which isco-owned by the present assignee, relates to IGFBP-4 amino acidsequences; copending application Ser. No. 07/577,392, filed Aug. 31,1990, which is co-owned by the present assignee, relates to geneticmaterial encoding IGFBP-4.

Zapf, J. et al., J. of Biol. Chem. (1990) 265:14892-14898, pertains tofour IGFBP's (IGFBP-2, IGFBP-3, a truncated form of IGFBP-3, andIGFBP-4) isolated from adult human serum by insulin-like growth factor(IGF) affinity chromatography and high performance liquidchromatography.

The existence of a number of different IGF-binding proteins indicatesthat these proteins may have different functions. Because it is possibleto diagnose disease states and to modify in various different ways thebiological activity of IGFs using the currently known binding proteins,there is significant interest, in the discovery of new IGF-bindingproteins having the same or different biological properties.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide an IGFbinding protein that differs from IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4,and IGFBP-6.

It is further an object of the present invention to provide a new IGFbinding protein using recombinant DNA molecules capable of expressingthe new IGF binding protein (designated herein as IGFBP-5) in order toproduce the binding protein.

These and other objects of the invention have been accomplished byproviding a purified IGFBP selected from a group consisting of an IGFBPhaving an amino acid sequence which is at least 60%, preferably 70% andmore preferably 85%, and most preferably 90%, homologous to the aminoacid sequence of FIG. 1 and fragments thereof wherein the fragments areof a sufficient length to be unique to this binding protein (e.g., 10,15, 20, or 25 consecutive amino acids of said sequence), and furtherwherein the purified binding protein is capable of binding to anantibody specific for IGFBP-5 or an insulin-like growth factor.Recombinantly produced binding protein molecules and antibodies thatrecognize the new binding protein are also part of the invention.

A significant advantage of producing IGFIBP-5 by recombinant DNAtechniques rather than by isolating IGFBP-5 from natural sources is thatequivalent quantities of IGFBP-5 can be produced by using less startingmaterial than would be required for isolating the binding protein from anatural source. Producing IGFBP-5 by recombinant techniques also permitsIGFBP-5 to be isolated in the absence of some molecules normally presentin cells that naturally produce IGFBP-5. Indeed, IGFBP compositionsentirely free of any trace of human protein contaminants can readily beproduced since the only human protein produced by the recombinantnon-human host is the recombinant IGFBP. Potential viral agents fromnatural sources are also avoided. It is also apparent that recombinantDNA techniques can be used to produce IGFBP-5 polypeptide derivativesthat are not found in nature, such as the variations described above.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic diagram showing amino acid (SEQ ID NO: 8) andnucleotide (SEQ ID NO: 7) sequences of a clone encoding human IGFBP-5.

DESCRIPTION OF SPECIFIC EMBODIMENTS

Novel compositions comprising recombinant proteins produced usingsequences encoding IGFBP-5 and fragments derived thereof are provided,together with proteins isolated from natural sources as well as proteinsexpressed recombinantly, and methods for producing these proteins.

1. Definitions

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See e.g.,Sambrook, et al., MOLECULAR CLONING; A LABORATORY MANUAL, SECOND EDITION(1989); DNA CLONING, VOLUMES I AND II (D. N Glover ed. 1985);OLIGONUCLEOTIDE SYNTHESIS (M. J. Gait ed, 1984); NUCLEIC ACIDHYBRIDIZATION (B. D. Hames & S. J. Higgins eds. 1984); TRANSCRIPTION ANDTRANSLATION (B. D. Hames & S. J. Higgins eds. 1984); ANIMAL CELL CULTURE(R. I. Freshney ed. 1986); IMMOBILIZED CELLS AND ENZYMES (IRL Press,1986); B. Perbal, A PRACTICAL GUIDE TO MOLECULAR CLONING (1984); theseries, METHODS IN ENZYMOLOGY (Academic Press, Inc.); GENE TRANSFERVECTORS FOR MAMMALIAN CELLS (J. H. Miller and M. P. Calos eds. 1987,Cold Spring Harbor Laboratory), Methods in Enzymology Vol. 154 and Vol.155 (Wu and Grossman, and Wu, eds., respectively), Mayer and Walker,eds. (1987), IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY(Academic Press, London), Scopes, (1987), PROTEIN PURIFICATION:PRINCIPLES AND PRACTICE, Second Edition (Springer-Verlag, N.Y.), andHANDBOOK OF EXPERIMENTAL IMMUNOLOGY, VOLUMES I-IV (D. M. Weir and C. C.Blackwell eds 1986).

Standard abbreviations for nucleotides and amino acids are used in FIG.1 and elsewhere in this specification.

As used herein, the term IGFBP-5 is an acronym for insulin-like growthfactor binding protein-5. This protein, or a fragment thereof, iscapable of binding to an antibody specific for IGFBP-5 or to an IGFfactor. A cDNA encoding at least one form of IGFBP-5 is presented in antFIG. 1. It is anticipated that other species of IGFBP-5 exist or thatthey can be created. Thus, IGFBP-5 refers to any of the naturallyoccurring forms of IGFBP-5, including the form shown in FIG. 1. In thesequence shown, the cleavage site for the mature protein may occur whereindicated by arrow (a), resulting in a protein having a molecular weightof 29,018 Da. Additionally, another species of the protein may becleaved where indicated by arrow (b), resulting in a protein having amolecular weight of approximately 28,500 Da.

Additionally, analogs are included within the definition and includetruncated polypeptides (including fragments) and IGFBP-5-likepolypeptides, e.g., mutants, that retain catalytic activity andpreferably have a homology of at least 80%, more preferably 90%, andmost preferably 95%. Typically, such analogs differ by only 1, 2, 3, or4 codon changes. Examples include polypeptides with minor amino acidvariations from the natural amino acid sequence of IGFBP-5; inparticular, conservative amino acid replacements. Conservativereplacements are those that take place within a family of amino acidsthat are related in their side chains. Genetically encoded amino acidsare generally divided into four families: (1) acidic=aspartate,glutamate; (2) basic=lysine, arginine, histidine; (3) non-polar=alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine,cystine, serine, threonine, tyrosine. Phenylalanine, tryptophan, andtyrosine are sometimes classified jointly as aromatic amino acids. Forexample, it is reasonable to expect that an isolated replacement of aleucine with an isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar conservative replacement of anamino acid with a structurally related amino acid will not have a majoreffect on the biological activity. Polypeptide molecules havingsubstantially the same amino acid sequence as IGFBP-5 but possessingminor amino acid substitutions that do not substantially affect theability of the IGFBP-5 polypeptide derivatives to interact withIGFBP-5-specific molecules, such as antibodies and IGF molecules,particularly IGF-I and especially IGF-II, are within the definition ofIGFBP-6. Derivatives include aggregative conjugates with other IGF-BPmolecules and covalent conjugates with unrelated chemical moieties.Covalent derivatives are prepared by linkage of functionalities togroups which are found in IGF-BP amino acid chains or at the N- orC-terminal residues by means known in the art.

IGFBP-5-specific molecules include polypeptides such as antibodies thatare specific for the IGFBP-5 polypeptide containing the naturallyoccurring IGFBP-5 amino acid sequence. By “specific binding polypeptide”is intended polypeptides that bind with IGFBP-5 and its derivatives andwhich have a measurably higher binding affinity for the targetpolypeptide, i.e., IGFBP-5 and polypeptide derivatives of IGFBP-5, thanfor other polypeptides tested for binding. Higher affinity by a factorof 10 is preferred, more preferably a factor of 100. Binding affinityfor antibodies refers to a single binding event (i.e., monovalentbinding of an antibody molecule). Specific binding by antibodies alsomeans that binding takes place at the normal binding site of themolecule's antibody (at the end of the arms in the variable region).

Utilizing the sequence data in FIG. 1, as well as the denotedcharacteristics of IGFBP-5, it is within the skill of the art to obtainother DNA sequences encoding IGFBP-5. For example, the structural genemay be manipulated by varying individual nucleotides, while retainingthe correct amino acid(s), or varying the nucleotides, so as to modifythe amino acids, without loss of biological activity. Nucleotides may besubstituted, inserted, or deleted by known techniques, including, forexample, in vitro mutagenesis and primer repair. The structural gene maybe truncated at its 3′-terminus and/or its 5′-terminus while retainingits biological activity.

The term “recombinant polynucleotide” as used herein intends apolynucleotide of genomic, cDNA, semisynthetic, or synthetic originwhich, by virtue of its origin or manipulation: (1) is not associatedwith all or a portion of a polynucleotide with which it is associated innature, (2) is linked to a polynucleotide other than that to which it islinked in nature, or (3) does not occur in nature.

The term “polynucleotide” as used herein refers to a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides. This term refers only to the primary structure ofthe molecule. Thus, this term includes double- and single-stranded DNAand RNA. It also includes known types of modifications, for example,labels which are known in the art, methylation, “caps”, substitution ofone or more of the naturally occurring nucleotides with an analog,internucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example proteins (including for e.g., nucleases, toxins, antibodies,signal peptides, poly-L-lysine, etc.), those with intercalators (e.g.,acridine, psoralen, etc.), those containing chelators (e.g., metals,radioactive metals, boron, oxidative metals, etc.)., those containingalkylators, those with modified linkages (e.g., alpha anomeric nucleicacids, etc.), as well as unmodified forms of the polynucleotide.

A “replicon” is any genetic element, e.g., a plasmid, a chromosome, avirus, a cosmid, etc. that behaves as an autonomous unit ofpolynucleotide replication within a cell; i.e., capable of replicationunder its own control. This may include selectable markers.

A “vector” is a replicon in which another polynucleotide segment isattached, so as to bring about the replication and/or expression of theattached segment.

“Control sequence” refers to polynucleotide sequences which arenecessary to effect the expression of coding sequences to which they areligated. The nature of such control sequences differs depending upon thehost organism; in prokaryotes, such control sequences generally includepromoter, ribosomal binding site, and transcription terminationsequence; in eukaryotes, generally, such control sequences includepromoters and transcription termination sequence. The term “controlsequences” is intended to include, at a minimum, all components whosepresence is necessary for expression, and may also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

Operably linked” refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. A control sequence “operably linked” to a codingsequence is ligated in such a way that expression of the coding sequenceis achieved under conditions compatible with the control sequences.

An “open reading frame” (ORF) is a region of a polynucleotide sequencewhich encodes a polypeptide; this region may represent a portion of acoding sequence or a total coding sequence.

A “coding sequence” is a polynucleotide sequence which is translatedinto a polypeptide, usually via mRNA, when placed under the control ofappropriate regulatory sequences. The boundaries of the coding sequenceare determined by a translation start codon at the 5′-terminus and atranslation stop codon at the 3′-terminus. A coding sequence caninclude, but is not limited to, cDNA, and recombinant polynucleotidesequences.

“PCR” refers to the technique of polymerase chain reaction as describedin Saiki, et al., Nature 324:163 (1986); U.S. Pat. No. 4,683,195; andU.S. Pat. No. 4,683,202.

As used herein, x is “heterologous” with respect to y if x is notnaturally associated with y in the identical manner; i.e., x is notassociated with y in nature or x is not associated with y in the samemanner as is found in nature.

“Homology” refers to the degree of similarity between x and y. It isexpected that the overall homology between different species or forms ofIGFBP-5 at the nucleotide level probably will be about 40% or greater,probably about 60% or greater, and even more probably about 80% to about90% or greater. The correspondence between the sequence from one form toanother can be determined by techniques known in the art. For example,they can be determined by a direct comparison of the sequenceinformation of the polynucleotide. Alternatively, homology can bedetermined by hybridization of the polynucleotides under conditionswhich form stable duplexes between homologous regions (for example,those which would be used prior to S₁ digestion), followed by digestionwith single-stranded specific nuclease(s), followed by sizedetermination of the digested fragments.

As used herein, the term “polypeptide” refers to a polymer of aminoacids and does not refer to a specific length of the product; thus,peptides, oligopeptides, and proteins are included within the definitionof polypeptide. This term also does not refer to or excludepost-expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like. Includedwithin the definition are, for example, polypeptides containing one ormore analogs of an amino acid (including, for example, unnatural aminoacids, etc.), polypeptides with substituted linkages, as well as othermodifications known in the art, both naturally occurring andnon-naturally occurring.

A polypeptide or amino acid sequence “derived from” a designated nucleicacid sequence refers to a polypeptide having an amino acid sequenceidentical to that of a polypeptide encoded in the sequence, or a portionthereof wherein the portion.consists of at least 3-5 amino acids, andmore preferably at least 8-10 amino acids, and even more preferably atleast 11-15 amino acids, or which is immunologically identifiable with apolypeptide encoded in the sequence. This terminology also includes apolypeptide expressed from a designated nucleic acid sequence.

IGFBP-5, or polypeptide derivatives thereof, may be used for producingantibodies, either monoclonal or polyclonal, specific to IGFBP-5. Theseterms, and the methods for producing antibodies are known in the art.

“Recombinant host cells”, “host cells”, “cells”, “cell lines”, “cellcultures”, and other such terms denote, for example, microorganisms,insect cells, and mammalian cells, that can be, or have been, used asrecipients for recombinant vector or other transfer DNA, and include theprogeny of the original cell which has been transformed. It isunderstood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement as the original parent, due to natural, accidental, ordeliberate mutation.

As used herein, the term “microorganism” includes prokaryotic andeukaryotic microbial species such as bacteria and fungi, the latterincluding yeast and filamentous fungi.

“Transformation”, as used herein, refers to the insertion of anexogenous polynucleotide into a host cell, irrespective of the methodused for the insertion, for example, direct uptake, transduction,f-mating or electroporation. The exogenous polynucleotide may bemaintained as a non-integrated vector, for example, a plasmid, oralternatively, may be integrated into the host genome.

By “purified” and “isolated” is meant, when referring to a polypeptideor nucleotide sequence, that the indicated molecule is present in thesubstantial absence of other biological macromolecules of the same type.The term “purified” as used herein preferably means at least 95% byweight, more preferably at least 99% by weight, and most preferably atleast 99.8% by weight, of biological macromolecules of the same typepresent (but water, buffers, and other small molecules, especiallymolecules having a molecular weight of less than 1000, can be present).

2. Specific Modes for Carrying Out the Invention

a. Sources of IGFBP-5

IGFBP-5 is derivable from mammals, e.g. murine, porcine, equine, bovine,and human sources. All such sources are included within the definitionof IGF-BP-5, as long as they comply with the required degree ofhomology.

IGF-BP-5 includes binding proteins purified from a tissue extract orfrom a conditioned culture medium as well as those obtained byrecombinant means.

b. Purification of IGFBP-5

IGFBP-5 can be readily purified from blood and its components, such asserum and plasma and from cells genetically modified to produce IGFBP-5or polypeptide derivatives thereof, by affinity chromatography using amonoclonal antibody specific for IGFBP-5. In addition to the use ofantibody affinity chromatography, IGFBP-5 and polypeptide derivativesthereof can be purified by a variety of other widely known proteinpurification techniques (either alone or in combination) includingimmunoprecipitation, gel filtration, ion exchange chromatography,chromatofocusing, isoelectric focusing, selective precipitation,electrophoresis, and the like. Fractions isolated during purificationprocedures can be analyzed for the presence of IGFBP-5 or polypeptidederivatives of IGFBP-5 by immunoassays employing IGFBP-5-specificantibodies or IGFBP-5-specific bioassays. Detailed examples are providedbelow.

c. Isolation of IGFBP-5 Sequences

Isolation of nucleotide sequences encoding IGFBP-5 involves creation ofeither a genomic library prepared from cells encoding IGFBP-5 orpreparation of a cDNA library from RNA isolated from cells expressingIGFBP-6. It will generally be preferable to create a cDNA library forisolation of IGFBP-5 coding nucleotide sequences so as to avoid anypossible problems arising from attempts to determine intron/exonborders. Genetic libraries can be made in either eukaryotic orprokaryotic host cells. Widely available cloning vectors such asplasmids, cosmids, phage, YACs and the like can be used to generategenetic libraries suitable for the isolation of nucleotide sequencesencoding IGFBP-5 or portions thereof.

d. Screening for the Presence of IGFBP-5 Sequences

Useful methods for screening genetic libraries for the presence ofIGFBP-5 nucleotide sequences include the preparation of oligonucleotideprobes based on the N-terminus amino acid sequence information frompurified IGFBP-5 or purified internal fragments of purified IGFBP-5. Byemploying the standard triplet genetic code, oligonucleotide sequencesof about 17 base pairs or longer can be prepared by conventional invitro synthesis techniques so as to correspond to portions of IGFBP-5for which the amino acid sequence has been determined by N-terminusanalysis. The resultant nucleic acid sequences can be subsequentlylabeled with radionuclides, enzymes, biotin, fluorescers, or the like,and used as probes for screening genetic libraries.

Additional methods of interest for isolating IGFBP-5-encoding nucleicacid sequences include screening genetic libraries for the expression ofIGFBP-5 or fragments thereof by means of IGFBP-5-specific antibodies,either polyclonal or monoclonal. A preferred technique involves the useof degenerate primers based on partial amino acid sequences of purifiedIGFBP-5 or on sequences from known related molecules and the polymerasechain reaction (PCR) to amplify gene segments between the primers. Thegene can then be isolated using a specific hybridization probe based onthe amplified gene segment, which is then analyzed for appropriateexpression of protein. A detailed description of this technique is setforth in the examples that follow.

e. Sequencing Methods

Nucleotide sequences encoding IGFBP-5 can be obtained from recombinantDNA molecules recovered, from IGFBP-5 genetic library isolates. Thenucleotide sequence encoding IGFBP-5 can be obtained by sequencing thenon-vector nucleotide sequences of these recombinant molecules.Nucleotide sequence information can be obtained by employing widely usedDNA sequencing protocols, such as Maxim and Gilbert sequencing, dideoxynucleotide sequencing, and the like. Examples of suitable nucleotidesequencing protocols can be found in Berger and Kimmel, Methods inEnzymology Vol. 52, Guide to Molecular Cloning Techniques, (1987)Academic Press. Nucleotide sequence information from several recombinantDNA isolates, including isolates from both cDNA and genomic libraries,may be combined so as to provide the entire amino acid coding sequenceof IGFBP-5 as well as the nucleotide sequences of introns within theIGFBP-5 gene, upstream nucleotide sequences, and downstream nucleotidesequences.

Nucleotide sequences obtained from sequencing IGFBP-5 specific geneticlibrary isolates are subjected to analysis in order to identify regionsof interest in the IGFBP-5 gene. These regions of interest include openreading frames, introns, promoter sequences, termination sequences, andthe like. Analysis of nucleotide sequence information is preferablyperformed by computer. Software suitable for analyzing nucleotidesequences for regions of interest is commercially available andincludes, for example, DNASIS™ (LKB). It is also of interest to useamino acid sequence information obtained from the N-terminus sequencingof purified IGFBP-5 when analyzing IGFBP-5 nucleotide sequenceinformation so as to improve the accuracy of the nucleotide sequenceanalysis.

f. Expression Systems

IGFBP-5 and polypeptide derivatives of IGFBP-6 can be expressed byrecombinant techniques when a DNA sequence encoding the relevantmolecule is functionally inserted into a vector. By “functionallyinserted” is meant in proper reading frame and orientation, as is wellunderstood by those skilled in the art. When producing a geneticconstruction containing a complete IGFBP-5 reading frame, a preferredstarting material is a cDNA library isolate encoding IGFBP-5. Typically,the IGFBP-5 gene will be inserted downstream from a promoter and will befollowed by a stop codon, although production as a hybrid proteinfollowed by cleavage may be used, if desired. In general,host-cell-specific sequences improving the production yield of IGFBP-5and IGFBP-6 polypeptide derivatives will be used and appropriate controlsequences will be added to the expression vector, such as enhancersequences, polyadenylation sequences, and ribosome binding sites.

i. Mammalian Systems

Once the appropriate coding sequence is isolated, it can be expressed ina variety of different expression systems; for example those used withmammalian cells, baculoviruses, bacteria, and yeast.

Mammalian expression systems are known in the art. A mammalian promoteris any DNA sequence capable of binding mammalian RNA polymerase andinitiating, the downstream (3′) transcription of a coding sequence (e.g.structural gene) into mRNA. A promoter will have a transcriptioninitiating region, which is usually placed proximal to the 5′ end of thecoding sequence, and a TATA box, usually located 25-30 base pairs (bp)upstream of the transcription initiation site. The TATA box is thoughtto direct RNA polymerase II to begin RNA synthesis at the correct site.A mammalian promoter will also contain an upstream promoter element,typically located within 100 to 200 bp upstream of the TATA box. Anupstream promoter element determines the rate at which transcription isinitiated and can act in either orientation [Sambrook et al. (1989)“Expression of Cloned Genes in Mammalian Cells.” In Molecular Cloning: ALaboratory Manual, 2nd ed.].

Mammalian viral genes are often highly expressed and have a broad hostrange; therefore sequences encoding mammalian viral genes provideparticularly useful promoter sequences. Examples include the SV40 earlypromoter, mouse mammary tumor virus LTR promoter, adenovirus major latepromoter (Ad MLP), and herpes simplex virus promoter. In addition,sequences derived from non-viral genes, such as the murinemetallotheionein gene, also provide useful promoter sequences.Expression may be either constitutive or regulated (inducible),depending on the promoter can be induced with glucocorticoid inhormone-responsive cells.

The presence of an enhancer element (enhancer), combined with thepromoter elements described above, will typically increase expressionlevels. An enhancer is a regulatory DNA sequence that can stimulatetranscription up to 1000-fold when linked to homologous or heterologouspromoters, with synthesis beginning at the normal RNA start site.Enhancers are also active when they are placed upstream or downstreamfrom the transcription initiation site, in either normal or flippedorientation, or at a distance of more than 1000 nucleotides from thepromoter [Maniatis et al. (1987) Science 236:1237; Alberts et al. (1989)Molecular Biology of the Cell, 2nd ed.]. Enhancer elements derived fromviruses may be particularly useful, because they typically have abroader host range. Examples include the SV40 early gene enhancer[Dijkema et al (1985) EMBO J. 4:761] and the enhancer/promoters derivedfrom the long terminal repeat (LTR) of the Rous Sarcoma Virus [Gorman etal. (1982b) Proc. Natl. Acad. Sci. 79:6777] and from humancytomegalovirus [Boshart et al. (1985) Cell 41:521]. Additionally, someenhancers are regulatable and become active only in the presence of aninducer, such as a hormone or metal ion [Sassone-Corsi and Borelli(1986) Trends Genet. 2:215; Maniatis et al. (1987) Science 236:1237].

A DNA molecule may be expressed intracellularly in mammalian cells. Apromoter sequence may be directly linked with the DNA molecule, in whichcase the first amino acid at the N-terminus of the recombinant proteinwill always be a methionine, which is encoded by the ATG start codon. Ifdesired, the N-terminus may be cleaved from the protein by in vitroincubation with cyanogen bromide.

Alternatively, foreign proteins can also be secreted from the cell intothe growth media by creating chimeric DNA molecules that encode a fusionprotein comprised of a leader sequence fragment that provides forsecretion of the foreign protein in mammalian cells. Preferably, thereare processing sites encoded between the leader fragment and the foreigngene that can be cleaved either in vivo or in vitro. The leader sequencefragment typically encodes a signal peptide comprised of hydrophobicamino acids which direct the secretion of the protein from the cell. Theadenovirus triparite leader is an example of a leader sequence thatprovides for secretion of a foreign protein in mammalian cells.

Typically, transcription termination and polyadenylation sequencesrecognized by mammalian cells are regulatory regions located 3, to thetranslation stop codon and thus, together with the promoter elements,flank the coding sequence. The 3′ terminus of the mature mRNA is formedby site-specific post-transcriptional cleavage and polyadenylation[Birnstiel et al. (1985) Cell 41:349; Proudfoot and Whitelaw (1988)“Termination and 3′ end processing of eukaryotic RNA. In Transcription;and splicing (ed. B. D. Hames and D. M. Glover); Proudfoot (1989) TrendsBiochem. Sci. 14:105]. These sequences direct the transcription of anmRNA which can be translated into the polypeptide encoded by the DNA.Examples of transcription terminator/polyadenylation signals includethose derived from SV40 [Sambrook et al (1989) “Expression of clonedgenes in cultured mammalian cells.” In Molecular Cloning: A LaboratoryManual].

Some genes may be expressed more efficiently when introns (also calledintervening sequences) are present. Several cDNAs, however, have beenefficiently expressed from vectors that lack splicing signals (alsocalled splice donor and acceptor sites) [see e.g., Gething and Sambrook(1981) Nature 293:620]. Introns, are intervening noncoding sequenceswithin a coding sequence that contain splice donor and acceptor sites.They are removed by a process called “splicing,” followingpolyadenylation of the primary transcript [Nevins (1983) Annu. Rev.Biochem. 52:441; Green (1986) Annu. Rev. Genet. 20:671; Padgett et al.(1986) Annu. Rev. Biochem. 55:1119; Krainer and Maniatis (1988) “RNAsplicing.” In Transcription and splicina (ed. B. D. Hames and D. M.Glover)].

Typically, the above described components, comprising a promoter,polyadenylation signal, and transcription termination sequence are puttogether into expression constructs. Enhancers, introns with functionalsplice donor and acceptor sites, and leader sequences may also beincluded in an expression construct, if desired. Expression constructsare often maintained in a replicon, such as an extrachromosomal element(e.g., plasmids) capable of stable maintenance in a host, such asmammalian cells or bacteria. Mammalian replication systems include thosederived from animal viruses, which require transacting factors toreplicate. For example, plasmids containing the replication systems ofpapovaviruses, such as SV40 [Gluzman (1981) Cell 23:175) orpolyomavirus, replicate to extremely high copy number in the presence ofthe appropriate viral T antigen. Additional examples of mammalianreplicons include those derived from bovine papillomavirus andEpstein-Barr virus. Additionally, the replicon may have two replicatonsystems, thus allowing it to be maintained, for example, in mammaliancells for expression and in a procaryotic host for cloning andamplification. Examples of such mammalian-bacteria shuttle vectorsinclude pMT2 [Kaufman et al. (1989) Mol. Cell. Biol. 9:946 and PHEBO[Shimizu et al. (1986) Mol. Cell. Biol. 6:1074].

The transformation procedure used depends upon the host to betransformed. Methods for introduction of heterologous polynucleotidesinto mammalian cells are known in the art and include dextran-mediatedtransfection, calcium phosphate precipitation, polybrene mediatedtransfection, protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

Mammalian cell lines available as hosts for expression are known in theart and include many immortalized cell lines available from the AmericanType Culture Collection (ATCC), including but not limited to, Chinesehamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells,monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g.,Hep G2), and a number of other cell lines.

ii. Baculovirus Systems

The polynucleotide encoding IGFBP-5 can also be inserted into a suitableinsect expression vector, and is operably linked to the control elementswithin that vector. Vector construction employs techniques which areknown in the art.

Generally, the components of the expression system include a transfervector, usually a bacterial plasmid, which contains both a fragment ofthe baculovirus genome, and a convenient restriction site for insertionof the heterologous gene or genes to be expressed; a wild typebaculovirus with a sequence homologous to the baculovirus-specificfragment in the transfer vector (this allows for the homologousrecombination of the heterologous gene in to the baculovirus genome);and appropriate insect host cells and growth media.

After inserting the IGFBP-5 DNA sequence into the transfer vector, thevector and the wild type viral genome are transfected into an insecthost cell where the vector and viral genome are allowed to recombine.The packaged recombinant virus is expressed and recombinant plaques areidentified and purified. Materials and methods for baculovirus/insectcell expression systems are commercially available in kit form from,inter alia, Invitrogen, San Diego Calif. (“MaxBac” kit). Thesetechniques are generally known to those skilled in the art and fullydescribed in Summers and Smith, Texas Agricultural Experiment StationBulletin No. 1555 (1987) (hereinafter “Summers and Smith”), andincorporated by reference.

Prior to inserting the IGFBP-5 DNA sequence into the baculovirus genome,the above described components, comprising a promoter, leader (ifdesired), coding sequence of interest, and transcription terminationsequence, are typically assembled into an intermediate transplacementconstruct (transfer vector). This construct may contain a single geneand operably linked regulatory elements; multiple genes, each with itsowned set of operably linked regulatory elements; or multiple genes,regulated by the same set of regulatory elements. Intermediatetransplacement constructs are often maintained in a replicon, such as anextrachromosomal element (e.g., plasmids) capable of stable maintenancein a host, such as a bacterium. The replicon will have a replicationsystem, thus allowing it to be maintained in a suitable host for cloningand amplification.

Currently, the most commonly used transfer vector for introducingforeign genes into AcNPV is pAc373. Many other vectors, known to thoseof skill in the art, have also been designed. These include, forexample, pVL985 (which alters the polyhedrin start codon from ATG toATT, and which introduces a BamHI cloning site 32 basepairs downstreamfrom the ATT; see Luckow and Summers, Virology (1989) 17:31.

The plasmid usually also contains the polyhedrin polyadenylation signal(Miller et al. (1988) Ann. Rev. Microbiol., 42:177) and a procaryoticampicillin-resistance (amp) gene and origin of replication for selectionand propagation in E. coli.

Baculovirus transfer vectors usually contain a baculovirus promoter. Abaculovirus promoter is any DNA sequence capable of binding abaculovirus RNA polymerase and initiating the downstream (5′ to 3′)transcription of a coding sequence (e.g. structural gene) into mRNA. Apromoter will have a transcription initiation region which is usuallyplaced proximal to the 5′ end of the coding sequence. This transcriptioninitiation region typically includes an RNA polymerase binding site anda transcription initiation site. A baculovirus transfer vector may alsohave a second domain called an enhancer, which, if present, is usuallydistal to the structural gene. Expression may be either regulated orconstitutive.

Structural genes, abundantly transcribed at late times in a viralinfection cycle, provide particularly useful promoter sequences.Examples include sequences derived from the gene encoding the viralpolyhedron protein, Friesen et al., (1986) “The Regulation ofBaculovirus Gene Expression,” in: The Molecular Biology of Baculoviruses(ed. Walter Doerfler); E.P.O. Pub. Nos. 127,839 and 155,476; and thegene encoding the p10 protein Vlak et al., (1988), J. Gen. Virol.69:765.

DNA encoding suitable signal sequences can be derived from genes forsecreted insect or baculovirus proteins, such as the baculoviruspolyhedrin gene (Carbonell et al. (1988) Gene, 73:409). Alternatively,since the signals for mammalian cell posttranslational modifications(such as signal peptide cleavage, proteolytic cleavage, andphosphorylation) appear to be recognized by insect cells, and thesignals required for secretion and nuclear accumulation also appear tobe conserved between the invertebrate cells and vertebrate cells,leaders of non-insect origin, such as those derived from genes encodinghuman α-interferon, Maeda et al., (1985), Nature 315:592; humangastrin-releasing peptide, Lebacq-Verheyden et al., (1988), Molec. Cell.Biol. 8:3129; human IL-2, Smith et al., (1985) Proc. Nat'l Acad. Sci.USA, 82:8404; mouse IL-3, (Miyajima et al., (1987) Gene 58:273; andhuman glucocerebrosidase, Martin et al. (1988) DNA, 7:99, can also beused to provide for secretion in insects.

A recombinant polypeptide or polyprotein may be expressedintracellularly or, if it is expressed with the proper regulatorysequences, it can be secreted. Good intracellular expression of nonfusedforeign proteins usually requires heterologous genes that ideally have ashort leader sequence containing suitable translation initiation signalspreceding an ATG start signal. If desired, methionine at the N-terminusmay be cleaved from the mature protein by in vitro incubation withcyanogen bromide.

Alternatively, recombinant polyproteins or proteins which are notnaturally secreted can be secreted from the insect cell by creatingchimeric DNA molecules that encode a fusion protein comprised of aleader sequence fragment that provides for secretion of the foreignprotein in insects. The leader sequence fragment typically encodes asignal peptide comprised of hydrophobic amino acids which direct thetranslocation of the protein into the endoplasmic reticulum.

After insertion of the IGFBP-5 DNA sequence and/or the gene encoding theexpression product precursor, an insect cell host is co-transformed withthe heterologous DNA of the transfer vector and the genomic DNA of wildtype baculovirus—usually by co-transfection. The promoter andtranscription termination sequence of the construct will typicallycomprise a 2-5kb section of the baculovirus genome. Methods forintroducing heterologous DNA into the desired site in the baculovirusvirus are known in the art. (See Summers and Smith supra; Ju et al.(1987); Smith et al., Mol. Cell. Biol. (1983) 3:2156; and Luckow andSummers (1989)). For example, the insertion can be into a gene such asthe polyhedrin gene, by homologous double crossover recombination;insertion can also be into a restriction enzyme site engineered into thedesired baculovirus gene. Miller et al., (1989), Bioessays 4:91. The DNAsequence, when cloned in place of the polyhedrin gene in, the expressionvector, is flanked both 5′ and 3′ by polyhedrin-specific sequences andis positioned downstream of the polyhedrin promoter.

The newly formed baculovirus expression vector is subsequently packagedinto an infectious recombinant baculovirus. Homologous recombinationoccurs at low frequency (between about 1% and about 5%); thus, themajority of the virus produced after cotransfection is still wild-typevirus. Therefore, a method is necessary to identify recombinant viruses.The beauty of the expression system is a visual screen allowingrecombinant viruses to be distinguished. The polyhedrin protein, whichis produced by the native virus, is produced at very high levels in thenuclei of infected cells at late times after viral infection.Accumulated polyhedrin protein forms occlusion bodies that also containembedded particles. These occlusion bodies, up to 15 μm in size, arehighly refractile, giving them a bright shiny appearance that is readilyvisualized under the light microscope. Cells infected with recombinantviruses lack occlusion bodies. To distinguish recombinant virus fromwild-type virus, the transfection supernatant is plaqued onto amonolayer of insect cells by techniques known to those skilled in theart. Namely, the plaques are screened under the light microscope for thepresence (indicative of wild-type virus) or absence (indicative ofrecombinant virus) of occlusion bodies. “Current Protocols inMicrobiology” Vol. 2 (Ausubel et al. eds) at 16.8 (Supp. 10, 1990);Summers and Smith, supra; Miller et al. (1989).

Recombinant baculovirus expression vectors have been developed forinfection into several insect cells. For example, recombinantbaculoviruses have been developed for, inter alia: Aedes aegypti,Autographa californica, Bombyx mori, Drosophila melanogaster, Spodopterafrugiperda, and Trichoplusia ni (P.C.T. Pub. No. WO89/046699; Carbonellet al., (1985) J. Virol. 56:153; Wright (1986) Nature 321:718; Smith etal., (1983) Mol. Cell. Biol. 3:2156; and see generally, Fraser, et al.(1989) In Vitro Cell. Dev. Biol. 25:225).

Cells and cell culture media are commercially available for both directand fusion expression of heterologous polypeptides in abaculovirus/expression system; cell culture technology is generallyknown to those skilled in the art. See. e.g., Summers and Smith supra.

The modified insect cells may then be grown in an appropriate nutrientmedium, which allows for stable maintenance of the plasmid(s) present inthe modified insect host. Where the expression product gene is underinducible control, the host may be grown to high density, and expressioninduced. Alternatively, where expression is constitutive, the productwill be continuously expressed into the medium and the nutrient mediummust be continuously circulated, while removing the product of interestand augmenting depleted nutrients. The product may be purified by suchtechniques as chromatography, e.g., HPLC, affinity chromatography, ionexchange chromatography, etc.; electrophoresis; density gradientcentrifugation; solvent extraction, or the like. As appropriate, theproduct may be further purified, as required, so as to removesubstantially any insect proteins which are also secreted in the mediumor result from lysis of insect cells, so as to provide a product whichis at least substantially free of host debris, e.g., proteins, lipidsand polysaccharides.

In order to obtain IGFBP-5 expression, recombinant host cells derivedfrom the transformants are incubated under conditions which allowexpression of the recombinant IGFBP-5 encoding sequence. Theseconditions will vary, dependent upon the host cell selected. However,the conditions are readily ascertainable to those of ordinary skill inthe art, based upon what is known in the art.

iii. Bacterial Systems

Bacterial expression techniques are known in the art. A bacterialpromoter is any DNA sequence capable of binding bacterial RNA polymeraseand initiating the downstream (3″) transcription of a coding sequence(e.g. structural gene) into mRNA. A promoter will have a transcriptioninitiation region which is usually placed proximal to the 5′ end of thecoding sequence. This transcription initiation region typically includesan RNA polymerase binding site and a transcription initiation site. Abacterial promoter may also have a second domain called an operator,that may overlap an adjacent RNA polymerase binding site at which RNAsynthesis begins. The operator permits negative regulated (inducible)transcription, as a gene repressor protein may bind the operator andthereby inhibit transcription of a specific gene. Constitutiveexpression may occur in the absence of negative regulatory elements,such as the operator. In addition, positive regulation may be achievedby a gene activator protein binding sequence, which, if present isusually proximal (5′) to the RNA polymerase binding sequence. An exampleof a gene activator protein is the catabolite activator protein (CAP),which helps initiate transcription of the lac operon in Escherichia coli(E. coli) [Raibaud et al. (1984) Annu. Rev. Genet. 18:173]. Regulatedexpression may therefore be either positive or negative, thereby eitherenhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly usefulpromoter sequences. Examples include promoter sequences derived fromsugar metabolizing enzymes, such as galactose, lactose (lac) [Chang etal. (1977) Nature 198:1056), and maltose. Additional examples includepromoter sequences derived from biosynthetic enzymes such as tryptophan(tr) [Goeddel et al. (1980) Nuc. Acids Res. 8:4057; Yelverton et al.(1981) Nucl. Acids Res. 9:731; U.S. Pat. No. 4,738,921; EPO Pub. Nos.036 776 and 121 775]. The g-laotamase (bla) promoter system [Weissmann(1981) “The cloning of interferon and other mistakes.” In Interferon 3(ed. I. Gresser)], bacteriophage lambda PL [Shimatake et al. (1981)Nature 292:128) and T5 [U.S. Pat. No. 4,689,406] promoter systems alsoprovide useful promoter sequences.

In addition, synthetic promoters which do not occur in nature alsofunction as bacterial promoters. For example, transcription activationsequences of one bacterial or bacteriophage promoter may be joined withthe operon sequences of another bacterial or bacteriophage promoter,creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433]. Forexample, the tac promoter is a hybrid trp-lac promoter comprised of bothtrp promoter and lac operon sequences that is regulated by the lacrepressor [Amann et al. (1983) Gene 25:167; de Boer et al. (1983) Proc.Natl. Acad. Sci. 80:21]. Furthermore, a bacterial promoter can includenaturally occurring promoters of non-bacterial origin that have theability to bind bacterial RNA polymerase and initiate transcription. Anaturally occurring promoter of non-bacterial origin can also be coupledwith a compatible RNA polymerase to produce, high levels of expressionof some genes in prokaryotes. The bacteriophase T7 RNApolymerase/promoter system is an example of a coupled promoter system[Studier et al. (1986) J. Mol. Biol. 189:113; Tabor et al. (1985) ProcNatl. Acad. Sci. 82:1074]. In addition, a hybrid promoter can also becomprised of a bacteriophage promoter and an E. coli operator region(EPO Pub. No. 267 851).

In addition to a functioning promoter sequence, an efficient ribosomebinding site is also useful for the expression of foreign genes inprokaryotes. In E. coli, the ribosome binding site is called theShine-Dalgarno (SD) sequence and includes an initiation codon (ATG) anda sequence 3-9 nucleotides in length located 3-11 nucleotides upstreamof the initiation codon (Shine et al. (1975) Nature 254:34]. The SDsequence is thought to promote binding of mRNA to the ribosome by thepairing of bases between the SD sequence and the 3′ and of E. coli 16SrRNA [Steitz et al. (1979) “Genetic signals and nucleotide sequences inmessenger RNA.” In Biological Regulation and Development: GeneExpression (ed. R. F. Goldberger)]. To express eukaryotic genes andprokaryotic genes with weak ribosome-binding site [Sambrook et al.(1989) “Expression of cloned genes in Escherichia coli.” In MolecularCloning: A Laboratory Manual].

A DNA molecule may be expressed intracellularly. A promoter sequence maybe directly linked with the DNA molecule, in which case the first aminoacid at the N-terminus will always be a methionine, which is encoded bythe ATG start codon. If desired, methionine at the N-terminus may becleaved from the protein by in vitro incubation with cyanogen bromide orby either in vivo on in vitro incubation with a bacterial methionineN-terminal peptidase (EPO Pub. No. 219 237).

Fusion proteins provide an alternative to direct expression. Typically,a DNA sequence encoding the N-terminal portion of an endogenousbacterial protein, or other stable protein, is fused to the 5′ end ofheterologous coding sequences. Upon expression, this construct willprovide a fusion of the two amino acid sequences. For example, thebacteriophage lambda cell gene can be linked at the 5′ terminus of aforeign gene and expressed in bacteria. The resulting fusion proteinpreferably retains a site for a processing enzyme (factor Xa) to cleavethe bacteriophage protein from the foreign gene [Nagai et al. (1984)Nature 309:810]. Fusion proteins can also be made with sequences fromthe lacZ (Jia et al. (1987) Gene 60:197], trpE [Allen et al. (1987) J.Biotechnol. 5:93; Makoff et al. (1989) J. Gen. Microbiol. 135:11], andChey [EPO Pub. No. 324 647] genes. The DNA sequence at the junction ofthe two amino acid sequences may or may not encode a cleavable site.Another example is a ubiquitin fusion protein. Such a fusion protein ismade with the ubiquitin region that preferably retains a site for aprocessing enzyme (e.g. ubiquitin specific processing-protease) tocleave the ubiquitin from the foreign protein. Through this method,native foreign protein can be isolated (Miller et al. (1989)Bio/Technolopy 7:698].

Alternatively, foreign proteins can also be secreted from the cell bycreating chimeric DNA molecules that encode a fusion protein comprisedof a signal peptide sequence fragment that provides for secretion of theforeign protein in bacteria (U.S. Pat. No. 4,336,336]. The signalsequence fragment typically encodes a signal peptide comprised ofhydrophobic amino acids which direct the secretion of the protein fromthe cell. The protein is either secreted into the growth media(gram-positive bacteria) or into the periplasmic spece, located betweenthe inner and outer membrane of the cell (gram-negative bacteria).Preferably there are processing sites, which can be cleaved either invivo or in vitro encoded between the signal peptide fragment and theforeign gene.

DNA encoding suitable signal sequences can be derived from genes forsecreted bacterial proteins, such as the E. coli outer membrane proteingene (ompA) [Masui et al. (1983), in: Experimental Manipulation of GeneExpression; Ghrayeb et al. (1984) EMBO J. 3:2437] and the E. colialkaline phosphatase signal sequence (phoA) [Oka et al. (1985) Proc.Natl. Acad. Sci. 82:7212]. As an additional example, the signal sequenceof the alpha-amylase gene from various Bacilus strains can be used tosecrete heterologous proteins from B. subtilis [Palva et al. (1982)Proc. Natl. Acad. Sci. USA 79:5582; EPO Pub. No. 244 042].

Typically, transcription termination sequences recognized by bacteriaare regulatory regions located 3′ to the translation stop codon, andthus together with the promoter flank the coding sequence. Thesesequences direct the transcription of an mRNA which can be translatedinto the polypeptide encoded by the DNA. Transcription terminationsequences frequently include DNA sequences of about 50 nucleotidescapable of forming stem loop structures that aid in terminatingtranscription. Examples include transcription termination sequencesderived from genes with strong promoters, such as the trp gene in E.coli as well as other biosynthetic genes.

Typically, the above described components, comprising a promoter, signalsequence (if desired), coding sequence of interest, and transcriptiontermination sequence, are put together into expression constructs.Expression constructs are often maintained in a replicon, such as anextrachromosomal element (e.g., plasmids) capable of stable maintenancein a host, such as bacteria. The replicon will have a replicationsystem, thus allowing it to be maintained in a procaryotic host eitherfor expression or for cloning and amplification. In addition, a repliconmay be either a high or low copy number plasmid. A high copy numberplasmid will generally have a copy number ranging from about 5 to about200, and typically about 10 to about 150. A host containing a high copynumber plasmid will preferably contain at least about 10, and morepreferably at least about 20 plasmids. Either a high or low copy numbervector may be selected, depending upon the effect of the vector and theforeign protein on the host.

Alternatively, the expression constructs can be integrated into thebacterial genome with an integrating vector. Integrating vectorstypically contain at least one sequence homologous to the bacterialchromosome that allows the vector to integrate. Integrations appear toresult from recombinations between homologous DNA in the vector and thebactedrial chromosome. For example, integrating vectors constructed withDNA from various Bacillus strains integrate into the Bacillus chromosome(EPO Pub. No. 127 328). Integrating vectors may also be comprised ofbacteriophage or transposon sequences.

Typically, extrachromosomal and integrating expression constructs maycontain selectable markers to allow for the selection of bacterialstrains that have been transformed. Selectable markers can be expressedin the bacterial host and may include genes which render bacteriaresistant to drugs such as ampicillin, chloramphenicol, erythromycin,kanamycin (neomycin), and tetracycline [Davies et al. (1978) Annu.Rev.Microbiol. 32:469]. Selectable markers may also include biosyntheticgenes, such as those in the histidine, tryptophan, and leucinebiosynthetic pathways.

Alternatively, some of the above described components can be puttogether in transformation vectors. Transformation vectors are typicallycomprised of a selectable market that is either maintained in a repliconor developed into an integrating vector, as described above.

Expression and transformation vectors, either extra-chromosomalreplicons or integrating vectors, have been developed for transformationinto many bacteria. For example, expression vectors have been developedfor, inter alia, the following bacteria: Bacillus subtilis [Palva et al.(1982) Proc. Natl. Acad. Sci. USA 79:5582; EPO Pub. Nos. 036 259 and 063953; PCT WO 84/04541], Escherichia coli [Shimatake et al. (1981) Nature292:128; Amann et al. (1985) Gene 40:183; Studier et al. (1986) J. Mol.Biol. 189:113; EPO Pub. Nos. 036 776, 136 829 and 136 907],Streptococcus cremoris [Powell et al. (1988) Appl. Environ. Microbiol.54:655]; Streptococcus lividans [Powell et al. (1988) Appl. Environ.Microbiol. 54:655], Streptomyces lividans [U.S. Pat. No. 4,745,056].

Methods of introducing exogenous DNA into bacterial hosts are well-knownin the art, and typically include either the transformation of bacteriatreated with CaCl₂ or other agents, such as divalent cations and DMSO.DNA can also be introduced into bacterial cells by electroporation.Transformation procedures usually vary with the bacterial species to betransformed. See e.g., [Masson et al. (1989) FEMS Microbiol. Lett.60:273; Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EPO Pub.Nos. 036 259 and 063 953; PCT WO 84/04541, Bacillus], [Miller et al.(1988) Proc. Natl. Acad. Sci. 85:856; Wang et al. (1990) J. Bacteriol.172:949, Campylobacter], [Cohen et al. (1973) Proc. Natl. Acad. Sci.69:2110; Dower et al. (1988) Nucleic Acids Res. 16:6127; Kushner (1978);“An improved method for transformation of Escherichia coli withColE1-derived plasmids. In Genetic Engineering: Proceedings of theInternational Symposium on Genetic Engineering (eds. H. W. Boyer and S.Nicosia); Mandel et al. (1970) J. Mol. Biol. 53:159; Taketo (1988)Biochim. Biophys. Acta 949:318; Escherichia], [Chassy et al. (1987) FEMSMicrobiol. Lett. 44:173 Lactobacillus]; [Fiedler et al. (1988) Anal.Biochem 170:38, Pseudomonas); [Augustin et al. (1990) FEMS Microbiol.Lett. 66:203, Staphylococcus], [Barany et al. (1980) J. Bacteriol.144:698; Harlander (1987) “Transformation Streptococcus lactis byelectroporation, in: Streptococcal Genetics (ed. J. Ferretti and R.Curtiss III); Perry et al. (1981) Infec. Immun. 32:1295; Powell et al.(1988) Appl. Environ. Microbiol. 54:655; Somkuti et al. (1987) Proc. 4thEvr. Cone. Biotechnology 1:412, Streptococcus]. iv. Yeast Expression

Yeast expression systems are also known to one of ordinary skill in theart. A yeast promoter is any DNA sequence capable of binding yeast RNApolymerase and initiating the downstream (3′) transcription of a codingsequence (e.g. structural gene) into mRNA. A promoter will have atranscription initiation region which is usually placed proximal to the5′ end of the coding sequence. This transcription initiation regiontypically includes an RNA polymerase binding site (the “TATA Box”) and atranscription initiation site. A yeast promoter may,also have a seconddomain called an upstream activator sequence (UAS), which, if present,is usually distal to the structural gene. The UAS permits regulated(inducible) expression. Constitutive expression occurs in the absence ofa UAS. Regulated expression may be either positive or negative, therebyeither enhancing or reducing transcription.

Yeast is a fermenting organism with an active metabolic pathway,therefore sequences encoding enzymes in the metabolic pathway provideparticularly useful promoter sequences. Examples include alcoholdehydrogenase (ADH) (EPO Pub. No. 284 044), enolase, glucokinase,glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase(GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglyceratemutase, and pyruvate kinase (PyK) (EPO Pub. No. 329 203). The yeast PHO5gene, encoding acid phosphatase, also provides useful promoter sequences[Myanohara et al. (1983) Proc. Natl. Acad. Sci. USA 80:1].

In addition, synthetic promoters which do not occur in nature alsofunction as yeast promoters. For example, UAS sequences of one yeastpromoter may be joined with the transcription activation region ofanother yeast promoter, creating a synthetic hybrid promoter. Examplesof such hybrid promoters include the ADH regulatory sequence linked tothe GAP transcription activation region (U.S. Pat. Nos. 4,876,197 and4,880,734). Other examples of hybrid promoters include promoters whichconsist of the regulatory sequences of either the ADH2, GAL4, GAL10, ORPHO5 genes, combined with the transcriptional activation region of aglycolytic enzyme gene such as GAP or PyK (EPO Pub. No. 164 556).Furthermore, a yeast promoter can include naturally occurring promotersof non-yeast origin that have the ability to bind yeast RNA polymeraseand initiate transcription. Examples of such promoters include, interalia, (Cohen et al. (1980) Proc. Natl. Acad. Sci. USA 77:1078; Henikoffet al. (1981) Nature 283:835; Hollenberg et al. (1981) Curr. ToDicsMicrobiol. Immunol. 96:119; Hollenberg et al. (1979) “The Expression ofBacterial Antibiotic Resistance Genes i the Yeast Saccharomycescerevisiae,” in: Plasmids of Medical. Environmental and CommercialImportance (eds. K>N>Timmis and A. Puhler); Mercerau-Puigalon et al.(1980) Gene 11:163; Panthier et al. (1980) Curr. Genet. 2:109;].

A DNA molecule may be expressed intracellularly in yeast. A promotersequence may be directly linked with the DNA molecule, in which case thefirst amino acid at the N-terminus of the recombinant protein willalways be a methionine, which is encoded by the ATG start codon. Ifdesired, methionine at the N-terminus may be cleaved from the protein byin vitro incubation with cyanogen bromide.

Fusion proteins provide an alternative for yeast expression systems, aswell as in mammalian, baculovirus, and bacterial expression systems.Typically, a DNA sequence encoding the N-terminal portion of anendogenous yeast protein, or other stable protein, is fused to the 5′end of heterologous coding sequences. Upon expression, this constructwill provide a fusion of the two amino acid sequences. For example, theyeast or human superoxide dismutase (SOD) gene, can be linked at the 5′terminus of a foreign gene and expressed in yeast. The DNA sequence atthe junction of the two amino acid sequences may or may not encode acleavable site. See e.g., EP6 Pub. No. 196 056. Another example is aubiquitin fusion protein. Such a fusion protein is made with theubiquitin region that preferably retains a site for a processing enzyme(e.g. ubiquitin-specific processing protease) to cleave the ubiquitinfrom the foreign protein. Through this method, therefore, native foreignprotein can be isolated (see, e.g., PCT WO 88/024066). This system is apreferred system for producing IGFBP-5.

Alternatively, foreign proteins can also be secreted from the cell intothe growth media by creating chimeric DNA molecules that encode a fusionprotein comprised of a leader sequence fragment that provide forsecretion in yeast of the foreign protein. Preferably, there areprocessing sites encoded between the leader fragment and the foreigngene that can be cleaved either in vivo or in vitro. The leader sequencefragment typically encodes a signal peptide comprised of hydrophobicamino acids which direct the secretion of the protein from the cell.

DNA encoding suitable signal sequences can be derived from genes forsecreted yeast proteins, such as the yeast invertase gene (EPO Pub. No.012 873; JPO Pub. No. 62,096,086) and the A-factor gene (U.S. Pat. No.4,588,684). Alternatively, leaders of non-yeast origin, such as aninterferon leader, exist that also provide for secretion in yeast (EPOPub. No. 060 057).

A preferred class of secretion leaders are those that employ a fragmentof the yeast alpha-factor gene, which contains both a “pre” signalsequence, and a “pro” region. The types of alpha-factor fragments thatcan be employed include the full-length pre-pro alpha factor leader(about 83 amino acid residues) as well as truncated alpha-factor leaders(typically about 25 to about 50 amino acid residues) (U.S. Pat. Nos.4,546,083 and 4,870,008; EPO Pub. No. 324 274). Additional leadersemploying an alpha-factor leader fragment that provides for secretioninclude hybrid alpha-factor leaders made with a presequence of a firstyeast, but a pro-region from a second yeast alpha-factor. (See e.g., PCTWO 89/02463.)

Typically, transcription termination sequences recognized by yeast areregulatory regions located 3′ to the translation stop codon, and thustogether with the promoter flank the coding sequence. These sequencesdirect the transcription of an mRNA which can be translated into thepolypeptide encoded by the DNA. Examples of transcription terminatorsequence and other yeast-recognized termination sequences, such as thosecoding for glycolytic enzymes.

Typically, the above described components, comprising a promoter, leader(if desired), coding sequence of interest, and transcription terminationsequence, are put together into expression constructs. Expressionconstructs are often maintained in a replicon, such as anextrachromosomal element (e.g., plasmids) capable of stable maintenancein a host, such as yeast or bacteria. The replicon may have tworeplication systems, thus allowing it to be maintained, for example, inyeast for expression and in a procaryotic host for cloning andamplification. Examples of such yeast-bacteria shuttle vectors includeYEp24 [Botstein et al. (1979) Gene 8:17-24], pCl/1 [Brake et al. (1984)Proc. Natl. Acad. Sci USA 81:4642-4646], and YRp17 [Stinchcomb et al.(1982) J. Mol. Biol. 158:1573]. In addition, a replicon may be either ahigh or low copy number plasmid. A high copy number plasmid willgenerally have a copy number ranging from about 5 to about 200, andtypically about 10 to about 150. A host containing a high copy numberplasmid will preferably have at least about 10, and more preferably atleast about 20. Enter a high or low copy number vector may be selected,depending upon the effect of the vector and the foreign protein on thehost. See e.g., Brake et al., supra.

Alternatively, the expression constructs can be integrated into theyeast genome with an integrating vector. Integrating vectors typicallycontain at least one sequence homologous to a yeast chromosome thatallows the vector to integrate, and preferably contain two homologoussequences flanking the expression construct. Integrations appear toresult from recombinations between homologous DNA in the vector and theyeast chromosome [Orr-Weaver et al. (1983) Methods in Enzymol.101:228-245]. An integrating vector may be directed to a specific locusin yeast by selecting the appropriate homologous sequence for inclusionin the vector. See Orr-Weaver et al., supra. One or more expressionconstruct may integrate, possibly affecting levels of recombinantprotein produced [Rine et al. (1983) Proc. Natl. Acad. Sci. USA80:6750]. The chromosomal sequences included in the vector can occureither as a single segment in the vector, which results in theintegration of the entire vector, or two segments homologous to adjacentsegments in the chromosome and flanking the expression construct in thevector, which can result in the stable integration of only theexpression construct.

Typically, extrachromosomal and integrating expression constructs maycontain selectable markers to allow for the selection of yeast strainsthat have been transformed. Selectable markers may include biosyntheticgenes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2,TRP1, and ALG7, and the G418 resistance gene, which confer resistance inyeast cells to tunicamycin and G418, respectively. In addition, asuitable selectable marker may also provide yeast with the ability togrow in the presence of toxic compounds, such as metal. For example, thepresence of CUP1 allows yeast to grow in the presence of copper ions[Butt et al. (1987) Microbiol, Rev. 51:351].

Alternatively, some of the above described components can be puttogether into transformation vectors. Transformation vectors aretypically comprised of a selectable marker that is either maintained ina replicon or developed into an integrating vector, as described above.

Expression and transformation vectors, either extrachromosomal repliconsor integrating vectors, have been developed for transformation into manyyeasts. For example, expression vectors have been developed for, interalia, the following yeasts:Candida albicans [Kurtz, et al. (1986) Mol.Cell. Biol. 6:142], Candida maltosa [Kunze, et al. 91985) J. BasicMicrobiol. 25:141]. Hansenula polymorpha [Gleeson, et al. (1986) J. Gen.Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302],Kluyveromyces fragilis [Das, et al. (1984) J. Bacteriol. 158:1165],Kluyveromyces lactis [De Louvencourt et al. (1983) J. Bacteriol.154:737; Van den Berg et al. (1990) Bio/Technology 8:135], Pichiaguillerimondii [Kunze et al. (1985) J. Basic Microbiol. 25:141], Pichiapastoris [Cregg, et al. (1985) Mol. Cell. Biol. 5:3376; U.S. Pat. Nos.4,837,148 and 4,929,555], Saccharomyces cerevisiae [Hinnen et al. (1978)Proc. Natl. Acad. Sci. USA 75:1929; Ito et al. (1983) J. Bacteriol.153:163], Schizosaccharomyces pombe [Beach and Nurse (1981) Nature300:706], and Yarrowia lipolytica [Davidow, et al. (1985) Curr. Genet.10:380471 Gaillardin, et al. (1985) Curr. Genet. 10:49].

Methods of introducing exogenous DNA into yeast hosts are well-known inthe art, and typically include either the transformation of spheroplastsor of intact yeast cells treated with alkali cations. Transformationprocedures usually vary with the yeast species to be transformed. Seee.g., [Kurtz et al. (1986) Mol. Cell. Biol. 6:142; Kunze et al. 91985)J. Basic Microbiol. 25:141; Candida]; [Gleeson et al. 91986) J. Gen.Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302;Hansenula]; [Das et al. (1984) J. Bacteriol. 158:1165; De Louvencourt etal. (1983) J. Bacteriol. 154:1165; Van den Berg et al. (1990)Bio/Technoloyy 8:135; Kluyveromyces]; [Cregg et al. (1985) Mol. Cell.Biol. 5:3376; Kunze et al. (1985) J. Basic Microbiol. 25:141; U.S. Pat.Nos. 4,837,148 and 4,929,555; Pichia]; [Hinnen et al. (1978) Proc. Natl.Acad. Sci. USA 75;1929; Ito et al. (1983) J. Bacteriol. 153:163Saccharomyces]; [Beach and Nurse (1981) Nature 300:706;Schizosaccharomyces]; [Davidow et al. (1985) Curr. Genet. 10:39;Gaillardin et al. (1985) Curr. Genet. 10:49; Yarrowia].

g. Production of Antibodies to IGFBP-5

Antibodies specific for IGFBP-5 are produced by immunizing anappropriate vertebrate host, e.g., rabbit, with purified IGFBP-5 orpolypeptide derivatives of IGFBP-5, by themselves or in conjunction witha conventional adjuvant. Usually, two or more immunizations will beinvolved, and blood or spleen will be harvested a few days after thelast injection. For polyclonal antisera, the immunoglobulins can beprecipitated, isolated and purified by a variety of standard techniques,including affinity purification using IGFBP-5 attached to a solidsurface, such as a gel or beads in an affinity column. For monoclonalantibodies, the splenocytes normally will be fused with an immortalizedlymphocyte, e.g., a myeloid cell line, under selective conditions forhybridoma formation. The hybridomas can then be cloned under limitingdilution conditions and their supernatants screened for antibodieshaving the desired specificity. Techniques for producing antibodies arewell known in the literature and are exemplified by the publicationAntibodies: A Laboratory Manual (1988) eds. Harlow and Lane, Cold SpringHarbor Laboratories Press, and U.S. Pat. Nos. 4,381,292, 4,451,570, and4,618,577.

For both in vivo use of antibodies to IGFBP-5 and anti-idiotypeantibodies and diagnostic use, it may be preferable to use monoclonalantibodies. Monoclonal anti-virus particle antibodies or anti-idiotypeantibodies can be produced as follows. The spleen or lymphocytes from animmunized animal are removed and immortalized or used to preparehybridomas by methods known to those skilled in the art. To produce ahuman-human hybridoma, a human lymphocyte donor is selected.Epstein-Barr virus (EBV) can be used to immortalize human lymphocytes ora human fusion partner can be used to produce human-human hybridomas.Primary in vitro immunization with peptides can also be used in thegeneration of human monoclonal antibodies. Antibodies secreted by theimmortalized cells are screened to determine the clones that secreteantibodies of the desired specificity.

h. Diagnostic Methods Using Antigens, Genetic Material, of Antibodies

The compositions comprising antigens or antibodies of the presentinvention, as well as the genetic material, can be used in diagnosticassays. Among the biologically useful information that can be obtainedis excessive binding protein levels due to the presence of tumors, thatresult in increased production of either IGF or one of the IGFBP bindingproteins (since the binding proteins are produced in the presence ofexcess IGF). Additionally, a number of known disorders can be related toIGF concentrations. For example, some types of osteoporosis are relatedto IGF levels. Additionally, the binding proteins can be used in theidentification, production, and purification of recombinantly producedIGFs. Methods for detecting the presence of IGFBP-5 comprise analyzing abiological sample such as a blood sample, cerebrospinal fluid, or tumoror bone tissue.

Typically, methods for detecting analytes such as binding proteins ofthe invention are based on immunoassays. Such techniques are well knownand need not be described here in detail. Examples include bothheterogeneous and homogeneous immunoassay techniques. Both techniquesare based on the formation of an immunological complex between thebinding protein and a corresponding specific antibody. Heterogeneousassays for IGFBP-5 typically use a specific monoclonal or polyclonalantibody bound to a solid surface. Sandwich assays are increasinglypopular. Homogeneous assays, which are carried out in solution withoutthe presence of a solid phase, can also be used, for example bydetermining the difference in enzyme activity brought on by binding offree antibody to an enzyme-antigen conjugate. A number of suitableassays are disclosed in U.S. Pat. Nos. 3,817,837, 4,006,360, 3,996,345.

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, or filter material. Theseattachment methods generally include non-specific adsorption of theprotein to the support or covalent attachment of the protein, typicallythrough a free amine group, to a chemically reactive group on the solidsupport, such as an activate carboxyl, hydroxyl, or aldehyde group.

In a second diagnostic configuration, known as a homogeneous assay,antibody binding to an analyte produces some change in the reactionmedium which can be directly detected in the medium. Known general typesof homogeneous assays proposed heretofore include (a) spinlabeledreporters, where antibody binding to the antigen is detected by a changein reported mobility (broadening of the spin splitting peaks), (b)fluorescent reporters, where binding is detected by a change influorescence efficiency, (c) enzyme reporters, where antibody bindingeffects enzyme/substrate interactions, and (d) liposome-bound reporters,where binding leads to liposome lysis and release of encapsulatedreporter. The adaptation of these methods to the protein antigen of thepresent invention follows conventional methods for preparing homogeneousassay reagents.

i. Diagnostic Applications Using Genetic Probes

The genetic material of the invention can it-self be used in numerousassays as probes for genetic material present in naturally occurringmaterials. The analyte can be a nucleotide sequence which hybridizeswith a probe comprising a sequence of (usually) at least about 16consecutive nucleotides, usually 30 to 200 nucleotides, up tosubstantially the full sequence of the sequences shown above (cDNAsequences). The analyte can be RNA or cDNA. The sample is typically a asdescribed in the previous section. A positive result is generallycharacterized as identifying a genetic material comprising a sequence atleast about 70% homologous to a sequence of at least 12 consecutivenucleotides of the sequences given herein, usually at least about 80%homologous to at least about 60 consecutive nucleotides within thesequences, and may comprise a sequence substantially homologous to thefull-length sequences. In order to detect an analyte, where the analytehybridizes to a probe, the probe may contain a detectable label. Probesthat are particularly useful for detecting binding proteins are based onconserved regions of these proteins, particularly from amino acids18.1-191 (PNCD) and amino acids 212-215 (CWCV) of BP6. These amino acidsare highly conserved in all of the related IGF binding proteins. OnlyIGFBP-1 has a difference, a N for a D at position 191.

One method for amplification of target nucleic acids, for later analysisby hybridization assays, is known as the polymerase chain reaction orPCR technique. The PCR technique can be applied to detecting IGFBP-5 ofthe invention in suspected samples using oligonucleotide primers spacedapart from each other and based on the genetic sequence set forthherein. The primers are complementary to opposite strands of a doublestranded DNA molecule and are typically separated by from about 50 to450 nt or more (usually not more than 2000 nt). This method entailspreparing the specific oligonucleotide primers and then repeated cyclesof target DNA denaturation, primer binding, and extension with a DNApolymerase to obtain DNA fragments of the expected length based on theprimer spacing. Extension products generated from one primer serve asadditional target sequences for the other primer. The degree ofamplification of a target sequence is controlled by the number of cyclesthat are performed and is theoretically calculated by the simple formula2n where n is the number of cycles. Given that the average efficiencyper cycle ranges from about 65% to 85%, 25 cycles produce from 0.3 to4.8 million copies of the target sequence. The PCR method is describedin a number of publications, including Saiki et al., Science (1985)230:1350-1354; Saiki et al., Nature (1986) 324:163-166; and Scharf etal., Science (1986) 233:1076-1078. Also see U.S. Pat. Nos. 4,683,194;4,683,195; and 4,683,202.

The invention includes a specific diagnostic method for determination ofIGFBP-5, based on selective amplification of IGFBP-5-encoding DNAfragments. This method employs a pair of single-strand primers derivedfrom non-homologous regions of opposite strands of a DNA duplex fragmentselected from the sequences set forth in FIG. 1. These “primerfragments,” which form one aspect of the invention, are prepared fromIGFBP-5 fragments such as described above. The method follows theprocess for amplifying selected nucleic acid sequences as disclosed inU.S. Pat. No. 4,683,202, as discussed above.

j. Assay for Biological Properties of IGFBP-5

The property of binding to an insulin-like growth factor is one of thebiological activities of the proteins of the invention. These proteinsmay be conveniently tested in a binding assay using IGF-I [Rinderknecht,E. and Humbel,R. E., J. Biol. Chem. (1978) 253 2769] or IGF-II[Rinderknecht, E. and Humbel, R. E., FEBS (1978) 89: 283], preferablyIGF-II, in a labelled, e.g., iodinated form. For example, such an assaymay conveniently include performing a gel electrophoresis (SDS-PAGE) ofthe proteins of the invention, followed by a western blot of the gel,then incubating the blot in the presence of [¹²⁵I]IGF-I or II, washingthe blot to remove free IGF-I or -II, and detecting the radioactivity onthe blot.

k. Uses of IGFBP-5

Therapeutic applications of the binding proteins of the inventioninclude its use as a single therapeutic agent and its use in combinationwith an IGF, the latter use being preferred.

When used in combination with an IGF, a binding protein of the inventionis suitable for use in the indications above mentioned, primarily as agrowth inducing, tissue regenerating or would healing agent.

Accordingly, the invention provides:

i) use of a binding protein of the invention together with IGF in freeor fixed combination for stimulating the growth of a subject, tissue ororgan regeneration or wound healing, or

ii) a method of stimulating the growth of a subject, tissue or organregeneration or wound healing in a subject which comprises administeringa therapeutically effective amount of a binding protein of the inventiontogether with a therapeutically effective amount of an IGF to a patientin need of such treatment, or

iii) a pharmaceutical composition for stimulating the growth of asubject, tissue or organ regeneration or wound healing which comprises abinding protein of a invention together with an IGF and with apharmaceutically acceptable carrier or diluent, or

iv) a package containing separate until dose forms of a binding proteinof the invention and an IGF, together with instructions for mixing orconcomitant administration.

In association with an IGF, a binding protein of the invention is ofspecial interest for mediating chondrogenesis or hematopoieses. This maybe shown in the following tests A to C.

A) An IGF increases bone formation as indicated by e.g. an increasedincorporation of [3H]-proline into collagen and non-collagen proteins infetal rat calvaria. A synergistic effect occurs when an IGF is used inthe presence of a binding protein of the invention. Organ cultures ofrat calvaria are prepared by dissecting frontal and parietal bones from21-day old fetal rats, splitting along the sagittal suture and culturingaccording to the method of Kream et al. (Endocrinology (1985) 116. 296).A binding protein or IGF is added in doses from 10 to 200 ng ml ofcultures. When they are added to combination to each other the molarratio is 1:1. Culturing is effected for 24 to 48 hours. To quantitatethe incorporation of [3H]proline into collagenase-digestible protein andnon-collagen protein, bone homogenates are digested with bacterialcollagenase according to the method of Diegelman R. and Peterkofsky(Dev. Biol. (1972) 28:443) and modified by Kream et al. (Endocrinology(1985) 116:296).

B) An IGF decreases bone resorption as indicated by a decrease inrelease of [45]Ca from bone. A synergistic effect occurs when an IGF isused in the presence of a binding protein of the invention. The test iseffected according to the principles of Raisz (J. Clin. Invest. (1965)44:103). Pregnant rats are injected s.c. with [45]Ca on the eighteenthday of gestation. An IGF, alone or in the presence of a binding proteinof the invention, is injected at a dose of 10 ng to 200 ng per animal.The binding protein is added so that the molar ratio of IGF is 1:1. Onday nineteen, the animals are sacrified, the fetuses removed. Themineralized shafts of the radii and ulnae are dissected and placed inculture. Resorption is quantitated on the basis of release of [45]Cafrom the bone explants.

C) The IGF-binding proteins of the invention as well as otherIGF-binding proteins potentiate the erythropoietin-like effect of IGF-I.This may be, in particular, demonstrated by testing IGF-I, e.g. 10 ng/mlIGF-I, alone and in combination with the mature IGF binding protein ofFIG. 1, e.g. a 50 μml aliquot of a supernatant derived from a culture ofa CHO cell line expressing the mature IGF binding protein of FIG. 1, ina CFU-E assay as described in Fagg, B. Roitsch, C. A. Cell, Physiol.(1986) 126:1. Whereas the result obtained with IGF-binding protein aloneis not significantly different from the control, a synergistic effect ofthe combination is seen when compared to IGF-I alone.

Further, the mitogenic activity of an IGF combined with a bindingprotein of the invention may be tested as follows: The incorporation of[³H] methylthymidine into CCL 39 cells (Chinese hamster lungfibroblasts) in culture is measured as described by Plouet et al. Cell.Miol. (1984) 30:105. In this assay, cell line CCI 39 is seeded in aplate at 40 000 cells per well in 0.5 ml MEM culture medium (Gibco)containing 10% fetal. calf serum 0.1% penicillin, 0.4% streptomcyin and0.5% fungizone. After 72 hours incubation at 37° C. in an atmosphereloaded with 5% CO₂. Cells are washed with MEM medium in the absence offetal call serum and then cultured in his medium for 20 hours. At thisstage, the cell culture is confluent and an IGF or a binding protein orboth together are inoculated each at a dose of 10 ng to 200 ng culturemedium. When added together the molar ratio must be 1:1. The test sampleis incubated at 37° C. for 24 hours and then added with 1 mCi [³H]methylthymidine in 10 ml PBS. After 4 hours incubation the incorporationof methylthymidine is stopped washing cells with PBS. Cells are fixedwith 0.5 ml trichloroacetic acid (5%) for 30 min. washed with water andfinally lysed with 0.5 ml of NaOH 0.1M for 2 hours at 37° C. 0.5 ml oflysate is transferred into a scintillation flask and mixed with 3 ml ofscintillation liquid for measuring b-radioactivity. The binding proteinpotentiates the mitogenic activity of IGF although the radioactivitylevel that is measured when a binding protein is used alone is notsubstantially different from that of the control sample.

More particularly a binding protein of the invention, in combinationwith an IGF is useful a) for treating hypopituitarism. Laron-typedwarfism, osteoporosis, anemias especially complications following anchronic renal failure and liver or kidney deficiency, and b) forpromoting healing of wounds such as ulcers and burns or those occuringin accidental events or resulting from surgery.

For use in association with a binding protein of the invention. IGF ispreferably selected from IGF-I as described in Rinderknecht, E. andHumbel, R. E., J. Biol. Chem. (1978) 253:2769. IGF-II as described inRinderknecht, E. and Humbel, R. E., FEBS (1978) 89:283 and anyderivative or fragment of IGF-I and IGF-II having an insulin-like growthfactor activity. Most preferably, this is IGF-II.

For use in association with an IGF, a binding protein of the inventionis preferably a protein which is from 85% to 100% homologous with preIGF-BP or IGF-BP as shown in FIG. 1.

When not associated with IGFS, binding proteins of the invention havefurther therapeutic applications in any physiological disordersresulting from an excessive production of free IGF, e.g. IGF-producingcancers such as breast or kidney cancer, diabetic proliferativeretinopathy or abnormal growth of tall children with high serum level offree IGF.

Accordingly, the invention also provides:

(a) the use of a binding protein of the invention for treatingphysiological disorders resulting from an excessive production of freeIGF by a mammalian, for example human body, e.g. IGF-producing cancers,diabetic retinopthy or abnormal growth of tall subjects, or

(b) a method of treating physioligical disorders resulting from anexcessive production of free IGF, e.g. IGF-producing cancers, diabeticretinopathy or abnormal growth of a subject which comprisesadministering a therapeutically effective amount of a binding protein ofthe invention to a subject in need of such treatment, or

(c) a pharmaceutical composition for treating physiological disordersresulting from an excessive production of free IGF, e.g. IGF-producingcancers, diabetic retinopathy or abnormal growth of a subject whichcomprises a binding protein of the invention in association with apharmaceutically acceptable carrier or diluent, or

(iv) a method of delivering IGFs to specific organs or tissues based onthe differential binding properties of IGFBP-5, as indicated bybiological testing.

Fragments of mutated forms of the pre-IGF-BP or IGF-BP as shown in FIG.1 are of particular value for treating the physiological disordersresulting from an excessive production of free IGF in the human body.

A binding protein of the invention, alone or in combination with an IGF,may be administered by any conventional route suitable for peptides, orparticular enterally, e.g. in the form of tablets or capsules or,preferably parenterally, e.g. subcutaneously or intravenously in theform of injections of infusions. Further, it may be also used topically,e.g. in the form of ointments or suspensions when used, e.g. as a woundhealing agent.

For all the above indications the appropriate dosage will of course varydepending upon, for example, the nature and severity of the disorder tobe treated and the mode of administration. For example, satisfactoryresults may be obtained in the treatment of osteoporosis or anemia atdaily dosages from about to 0.1 mg/kg to 40 mg/kg body weight,preferably from about 0.5 mg/kg to about 20 mg/kg body weight of abinding protein of the invention. In larger mammals, for example humans,as indicated daily dosage is from about 5 mg conveniently administeredparenterally, for example once a day. For wound healing, a daily dose offrom 0.1 to 10 mg of a protein of the invention per cm² wound area issuitably indicated in larger mammals, for example humans. This isconveniently administered once a day. When used in combination with anIGF, the molar ratio of the binding protein to IGF is preferably from0.1:1 to 5:1, more preferably from 0.5:1 to 2:1, most preferably 1:1.

Pharmaceutical compositions of the invention may be manufactured inconventional manner.

Other uses for the binding proteins of the invention include varioususes in the production of IGF molecules by recombinant techniques. Thebinding proteins of the invention can be used to detect yeast-producedIGF in native (active) conformation (as opposed to inactivated forms).Additionally, the proteins of the invention can be used as carrier(possibly in the form of co-expressed proteins) in the production ofIGF. As the binding protein stabilized IGF in vivo, they are expected todo the same in vitro. The binding proteins can also be used to purifyIGF produced in yeast by attaching them to a solid surface (such as inaffinity chromatography).

Although the invention has been described with reference to particularembodiments, methods, construction, and use, it will be apparent tothose skilled in the art that various changes and modifications can bemade without departing from the invention.

3. Examples Tissues

Human osteosarcoma tissue was obtained from Dr. Marshall Urist (Univ. ofCalifornia, Los Angeles).

RNA Isolation

RNA was isolated by the guanidinium thiocyanate method [chirgivin, J. M.et al. (1979) Biochemistry 18: 5294-5299] with modification [Freeman, G.J. et al. (1983) Proc. Natl. Acad. Sci. USA 80: 4094-4098]. Poly (A)⁺RNA was purified by a single fractionation over oligo(dT)-cellulose[Aviv, H. and Leder, P. (1972) Proc. Natl. Acad. Sci USA 69: 1408-1412].

Oligonucleotide Synthesis

Oligonucleotide adapters, probes and primers were synthesized by thephosphoramidite method with an Applied Biosystems model 380Asynthesizer, purified by polyacrylamide gel electrophoresis and desaltedon Sep-Pak C₁₈ cartridges (Waters, Milford, Mass.).

A 14-mer oligonucleotide (5′ CCTGTAGATCTCCG 3′) (SEQ ID NO: 1) and18-mer oligonucleotide (5′ AATTCGGAGATCTACAGG 3′) (SEQ ID NO: 2) weresynthesized and used as the EcoRI adaptors for the cDNA libraryconstructed in λZAPII. The 14-mer was phosphorylated [Maniatis, T. etal. (1982). Molecular Cloning, a Laboratory Manual (Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.)], then immediately heated to 95°C. for 15 minutes to inactivate the polynucleotide kinase. The adaptorsalso contain an internal Bg1II site.

The two consensus PCR primers used to identify BP5 were a sense primerconsisting of a mixture of 32 24-mers (5′AGATCTGAATTCGCCXAA(C/T)TG(C/T)(A/G)A 3′) (SEQ ID NO: 3) and an sntisenseprimer consisting of a mixture of 16 25-mers (5′AGATCTAAGCTTCXAC(A/G)CACCA(A/G)CA 3′) (SEQ ID NO: 4) where X denotes allfour deoxynucleotides. EcoRI and HindIII sites were included in thesense and antisense primers, respectively, to allow for subcloning ofthe PCR products into M13 sequencing vectors.

The BP5 probes used to screen the cDNA library were two 19-mers, (5′GCAAAGGATTCTACAAGAG 3′) (SEQ ID NO: 5) and (5′ CAAACCTTCCCGTGGCCGC 3′)(SEQ ID NO: 6).

PCR Amplification

The PCR reactions were performed with the PCR kit (Perkin Elmer Cetus)according to the instructions of the supplier using the PCR primersdescribed above at a final concentration of 8 μM. The template cDNA wassynthesized from 2.5 μg of human osteosarcoma (Ost2) poly(A)⁺ RNA. Theconditions of cDNA synthesis were identical to those for first strandcDNA synthesis (see construction of cDNA library). The cDNA wasfractionated on Biogel A-15 m, recovered by ethanol precipitation andresuspended in 100 μl of sterile water. 1 μl of cDNA template was usedfor the PCR reaction. 35 cycles of PCR were performed in a Perkin ElmerCetus DNA thermal cycler. The first 10 cycles consisted of a 94C, 1minute denaturation step, a 33° C., 1 minute annealing step and a 33°C., 1 minute extension step. The next 25 cycles consisted of a 94° C., 1minute denaturation step, a 55° C., 1 minute annealing step and a 72°C., 1 minute extension step. The final extension step of the last cyclewas 7 minutes. The sample was extracted once withphenol/chloroform/isoamylalcohol (1:1:0.04), once withchloroform/isoamylalcohol (24:1) and recovered by ethanol precipitation.The PCR DNA product was then incubated for 20 minutes at 37° C. with 10units of DNA polymerase I, Klenow fragment in 10 mM Tris-HCl pH 7.5, 10mM MgCl₂, 50 mM NaCl, 1 mM ditheothreitol and 40 μM each of DATP, dGTP,dTTP and dCTP. The sample was extracted as above, recovered by ethanolprecipitation, digested with Eco RI and Hind III, and fractionated byelectrophoresis on a 7% acrylamide, 1×TBE (Tris/borate/EDTA) gel. DNAmigrating between 80-100 base pairs was excised from the gel; passivelyeluted from 16 hours with gentle shaking in 10 mM Tris-hydrochloride pH7.5, 1 mM EDTA, purified by passage over an elutip-D column as describedby the supplier (Schleicher and Schuell), ligated to an EcoRI andHindIII cut M13 sequencing vector (mp18) and introduced into E. colistrain DH5αF′.

Construction of the cDNA Library

A λZAPII/human osteosarcoma cDNA library was constructed from humanosteosarcoma poly(A)⁺ RNA as described in Zapf et al. (1990) J. Biol.Chem. 265: 14892-14898. A library of 1.75×10⁷ independent recombinantclones was obtained.

Screening of the cDNA Library

Approximately 300,000 recombinant phages from the Ost4 cDNA library wereplated (50,000 phages/137 mm diameter plate) in E.coli BB4 and grown for5-6 hours at 37° C. The phages were transferred onto nitrocellulosefilters (Millipore, HAFT 137), processed [Benton, W. D., and Davis, R.W. (1977) Science 196: 180-182] and screened with two IGFBP-5 probes.The probes were labeled with T₄ polynucleotide kinase and [γ³²P]ATP[Maniatis, T. et al. (1982) Molecular Cloning, A Laboratory Manual (ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.)] to a specificactivity of 1-2×10⁸ cpm/μg. The filters were prehybridized for 1-2 hoursat 37C as described in Zapf et al. (1990) J. Biol. Chem. 265:14892-14898. Labeled probe was added to a concentration of 10⁶ cpm/ml:and hybridization was continued overnight at 37° C. with gentle shaking.The filters were washed in 2×SSC (1×SSC=0.15M sodium chloride/0.015Msodium citrate, pH 7), 0.1% SDS at 50C and exposed overnight at −80° C.to Kodak XAR-2 films with a Du Pont Lightning Plus intensifying screen.Areas of plaques giving duplicate signals were picked, replicated andrescreened until pure plaques were obtained.

Plasmid Isolation, Subdloning and Sequencing

Bluescript SK(−) plasmids containing IGFBP-5 cDNA inserts were releasedfrom λZAP by the M13 rescue/excision protocol described by the supplier(Stratagene). Plasmid DNA was isolated by the alkaline lysis method[Maniatis, T. et al. (1982) Molecular Cloning, A Laboratory Manual:(Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.)]. The insertswere excised from the Bluescript SK(−) vector by BglII or EcoRIdigestion and fractionated by agarose gel electrophoresis. Inserts werecut out from the gel and eluted for 12 hours with gentle shaking in 10mM Tris-hydrochloride pH 7.5, 1 mM EDTA (TE), purified over an elutip-Dcolumn (see above) and subcloned into a M13 sequencing vector[Yanish-Perron, C. et al. (1985) Gene 33: 103-119]. DNA sequencing wasperformed by the dideoxy chain termination method [Sanger, F. et al.(1977) Proc. Natl. Acad. Sci. USA 74: 5463-5467] using M13 primers aswell as specific internal primer. Ambiguous regions were resolved using7-deaza-2-deoxyguanosine-triphosphate [Barr, P. J. et al. (1986)Biotechniques 4: 428-432] and Sequenase (US Biochemicals).

Northern Blot Analysis

Poly(A)⁺ RNA was fractionated on 1.4% agarose gel in the presence offormaldehyde [Lehrach, H. et al. (1977) Biochemistry 16: 4743-4751],transferred directly to nitrocellulose, and processed as described[Thomas, P. (1980) Proc. Natl. Acad. Sci. USA 77: 5201-5205]. Filterswere hybridized with the purified cDNA insert of BP6.1 as describedabove (screening of cDNA library). The filters were washed twice for 20minutes in 0.1×SSC, 0.1% SDS at 65C. The cDNA probes were labeled asdescribed [Thomas, P. (1980) Proc. Natl. Acad. Sci. USA 77: 5201-5205]to a specific activity of 2×10⁹ cpm/mg.

Results

Identification and Cloning of IGFBP-5

An amino acid sequence comparison of the five know human IGFBPs revealeda high degree of homology in the NH2- and the COOH—terminal regions. Thelongest stretch of identical amino acids in all five of the BPs residesin two areas of the COOH- terminal region consisting of three aminoacids Pro-Asn-Cys and four amino acids Cys-Trp-Cys-Val. These conservedamino acids fall within a region of the BPs that was shown to behomologous to 10 repeats within the amino terminal two thirds of thethyroglobulin molecule.

In an attempt to identify new BPs degenerate primers were desinged basedon these sequences and PCR was performed using human osteosarcoma cDNAas a template. DNA sequence analysis of the eight PCR products yieldedone sequence identical to BP2, three identical to BP4, three identicalto BP5 and one unique sequence, which was designated IGFBP5, and showeda 60% DNA sequence identity and a 76% amino acid identity to BP3.

Based on the PCR DNA sequence of IGFBP-5, two unique IGFBP-5 DNA probeswere synthesized and used to screen a λZAPII/human osteosarcoma cDNAlibrary. From the a 300,000 recombinant clones screened, twelve cloneswere identified which hybridized to both probes. Five clones werefurther purified and the cDNA inserts were analyzed by BglII and EcoRIrestriction enzyme digestion and agarose gel electrophoresis. The cDNAsfell into two size classes of approximately 1.7 kb and 6 kb, which areexemplified by clones 1 and 12, respectively.

Expression of IGFBP-5 mRNA

Northern blot analysis of several different tissues using ³²P-labeledclone 1 cDNA, confirmed that these two size classes of IGFBP-5 mRNAexisted and suggested that osteoblasts are the main source of IGFBP-5mRNA. All of the tissues tested (liver, kidney, and brain) producedIGFBP-5 mRNA but at lower levels.

Sequence Analysis of IGFBP-5

IGFBP-5 clone 1 (BP6.1) cDNA was sequenced and is shown in FIG. 1 withthe deduced amino acid sequence. The amino terminal region of IGFBP-5 ishydrophobic and is presumably a signal peptide. The predicted signalpeptidase cleavage site (↓a) [von Heye (1986) Nucleic Acids Research 11:4683-4690] follows amino acid 15, yielding a mature molecule of 257amino acids with a MW of 29,018 Da. There are no N-glycosylation sites.There are 18 cysteine residues in IGFBP-5 all of which coincide withcysteine residues in BPs 1-5. There is some degree of amino acidhomology between IGFBP-5 and the other five BPs, which is mostpronounced in the amino and the carboxyl terminal regions of themolecules.

4. Deposit of Biological Material

Escherichia coli strain HB101 host cells transformed with pBsBP6.1 havebeen deposited on Dec. 18, 1990, with the American Type CultureCollection (ATCC), 10801University Boulevard, Manassas, Va. and givenaccession number 68496. This deposit will be maintained under the termsof the Budapest Treaty on the International Recognition of the Depositof Micro-organisms for purposes of patent procedure. The accessionnumber is available from the ATCC.

This deposit is provided merely as convenience to those of skill in theart, and are not an admission that a deposit is required under 35 U.S.C.112. The nucleic acid sequence of this plasmid, as well as the aminoacid sequence of the polypeptide encoded thereby, are incorporatedherein by reference and are controlling in the event of any conflictwith the description herein. A license may be required to make, use, orsell the deposited material, and no such license is hereby granted.

All patents, patent applications, and references cited herein areincorporated by reference.

What is claimed is:
 1. A purified polypeptide capable of binding to aninsulin-like growth factor selected from the group consisting of humaninsulin-like growth factor I and human insulin-like growth factor II,said polypeptide selected from the group consisting of (a) a polypeptidecomprising an amino acid sequence that has at least 80% sequenceidentity to the amino acid sequence depicted at positions 1-272, of SEQID NO:8; (b) a polypeptide comprising an amino acid sequence that has atleast 80% sequence identity to the amino acid sequence depicted atpositions 16-272, of SEQ ID NO:8; (c) a polypeptide comprising an aminoacid sequence that has at least 80% sequence identity to the amino acidsequence depicted at positions 21-272, of SEQ ID NO:8; and (d) apolypeptide comprising a fragment of the amino acid sequence depicted atpositions 1-272 of SEQ ID NO:8 wherein said fragment comprises at least10 consecutive amino acids thereof.
 2. The polypeptide of claim 1,wherein the polypeptide comprises a polypeptide comprising an amino acidsequence that has at least 80% sequence identity to the amino acidsequence depicted at positions 1-272 of SEQ ID NO:8.
 3. The polypeptideof claim 1, wherein the polypeptide consists of an amino acid sequencethat has at least 80% sequence identity to the amino acid sequencedepicted at positions 1-272 of SEQ ID NO:8.
 4. The polypeptide of claim1, wherein the polypeptide consists of an amino acid sequence that hasat least 90% sequence identity to the amino acid sequence depicted atpositions 1-272 of SEQ ID NO:8.
 5. The polypeptide of claim 1, whereinthe polypeptide consists of an amino acid sequence that has at least 95%sequence identity to the amino acid sequence depicted at positions 1-272of SEQ ID NO:8.
 6. The polypeptide of claim 1, wherein the polypeptideconsists of the amino acid sequence depicted at positions 1-272 of SEQID NO:8.
 7. The polypeptide of claim 1, wherein the polypeptidecomprises the amino acid sequence depicted at positions 1-272 of SEQ IDNO:8.
 8. The polypeptide of claim 1, wherein the polypeptide comprises apolypeptide comprising an amino acid sequence that has at least 80%sequence identity to the amino acid sequence depicted at positions16-272 of SEQ ID NO:8.
 9. The polypeptide of claim 1, wherein thepolypeptide consists of an amino acid sequence that has at least 80%sequence identity to the amino acid sequence depicted at positions16-272 of SEQ ID NO:8.
 10. The polypeptide of claim 1, wherein thepolypeptide consists of an amino acid sequence that has at least 90%sequence identity to the amino acid sequence depicted at positions16-272 of SEQ ID NO:8.
 11. The polypeptide of claim 1, wherein thepolypeptide consists of an amino acid sequence that has at least 95%sequence identity to the amino acid sequence depicted at positions16-272 of SEQ ID NO:8.
 12. The polypeptide of claim 1, wherein thepolypeptide consists of the amino acid sequence depicted at positions16-272 of SEQ ID NO:8.
 13. The polypeptide of claim 1, wherein thepolypeptide comprises the amino acid sequence depicted at positions16-272 of SEQ ID NO:8.
 14. The polypeptide of claim 1, wherein thepolypeptide comprises a polypeptide comprising an amino acid sequencethat has at least 80% sequence identity to the amino acid sequencedepicted at positions 21-272 of SEQ ID NO:8.
 15. The polypeptide ofclaim 1, wherein the polypeptide consists of an amino acid sequence thathas at least 80% sequence identity to the amino acid sequence depictedat positions 21-272 of SEQ ID NO:8.
 16. The polypeptide of claim 1,wherein the polypeptide consists of an amino acid sequence that has atleast 90% sequence identity to the amino acid sequence depicted atpositions 21-272 of SEQ ID NO:8.
 17. The polypeptide of claim 1, whereinthe polypeptide consists of an amino acid sequence that has at least 95%sequence identity to the amino acid sequence depicted at positions21-272 of SEQ ID NO:8.
 18. The polypeptide of claim 1, wherein thepolypeptide consists of the amino acid sequence depicted at positions21-272 of SEQ ID NO:8.
 19. The polypeptide of claim 1, wherein thepolypeptide comprises the amino acid sequence depicted at positions21-272 of SEQ ID NO:8.
 20. The polypeptide of claim 1, wherein thepolypeptide comprises a fragment of the amino acid sequence depicted atpositions 1-272 of SEQ ID NO:8 and wherein said fragment comprises atleast 25 consecutive amino acids thereof.
 21. A composition comprising apharmaceutically acceptable carrier or diluent, and the purifiedpolypeptide of claim
 1. 22. A pharmaceutical composition for treating aphysiological disorder that results from an excessive production of freeIGF comprising: (a) the polypeptide of claim 1, and (b) apharmaceutically acceptable carrier or diluent.
 23. A polypeptideproduced by a process comprising the steps of (a) providing a host celltransformed by a nucleic acid sequence encoding a polypeptide capable ofbinding to an insulin-like growth factor selected from the groupconsisting of human insulin-like growth factor I and human insulin-likegrowth factor II, said polypeptide selected from the group consisting of(a) a polypeptide comprising an amino acid sequence that has at least80% sequence identity to the amino acid sequence depicted at positions1-272, of SEQ ID NO:8; (b) a polypeptide comprising an amino acidsequence that has at least 80% sequence identity to the amino acidsequence depicted at positions 16-272, of SEQ ID NO:8; (c) a polypeptidecomprising an amino acid sequence that has at least 80% sequenceidentity to the amino acid sequence depicted at positions 21-272, of SEQID NO:8; and (d) a polypeptide comprising a fragment of the amino acidsequence depicted at positions 1-272 of SEQ ID NO:8 wherein saidfragment comprises at least 10 consecutive amino acids thereof, whereinthe nucleic acid sequence is under the control of a promoter capable ofproviding for expression of the polypeptide in the host cell; and (b)culturing the host cell under conditions that permit expression of thepolypeptide.
 24. The polypeptide of claim 23, wherein said nucleic acidsequence encodes a polypeptide comprising an amino acid sequence thathas at least 80% sequence identity to the amino acid sequence depictedat positions 1-272 of SEQ ID NO:8.
 25. The polypeptide of claim 23,wherein said nucleic acid sequence encodes a polypeptide that consistsof an amino acid sequence that has at least 80% sequence identity to theamino acid sequence depicted at positions 1-272 of SEQ ID NO:8.
 26. Thepolypeptide of claim 23, wherein said nucleic acid sequence encodes apolypeptide that consists of an amino acid sequence that has at least90% sequence identity to the amino acid sequence depicted at positions1-272 of SEQ ID NO:8.
 27. The polypeptide of claim 23, wherein saidnucleic acid sequence encodes a polypeptide that consists of an aminoacid sequence that has at least 95% sequence identity to the amino acidsequence depicted at positions 1-272 of SEQ ID NO:8.
 28. The polypeptideof claim 23, wherein said nucleic acid sequence encodes a polypeptidethat consists of the amino acid sequence depicted at positions 1-272 ofSEQ ID NO:8.
 29. The polypeptide of claim 23, wherein said nucleic acidsequence encodes a polypeptide comprising the amino acid sequencedepicted at positions 1-272 of SEQ ID NO:8.
 30. The polypeptide of claim23, wherein said nucleic acid sequence encodes a polypeptide thatcomprises a polypeptide comprising an amino acid sequence that has atleast 80% sequence identity to the amino acid sequence depicted atpositions 16-272 of SEQ ID NO:8.
 31. The polypeptide of claim 23,wherein said nucleic acid sequence encodes a polypeptide that consistsof an amino acid sequence that has at least 80% sequence identity to theamino acid sequence depicted at positions 16-272 of SEQ ID NO:8.
 32. Thepolypeptide of claim 23, wherein said nucleic acid sequence encodes apolypeptide that consists of an amino acid sequence that has at least90% sequence identity to the amino acid sequence depicted at positions16-272 of SEQ ID NO:8.
 33. The polypeptide of claim 23, wherein saidnucleic acid sequence encodes a polypeptide that consists of an aminoacid sequence that has at least 95% sequence identity to the amino acidsequence depicted at positions 16-272 of SEQ ID NO:8.
 34. Thepolypeptide of claim 23, wherein said nucleic acid sequence encodes apolypeptide consists of the amino acid sequence depicted at positions16-272 of SEQ ID NO:8.
 35. The polypeptide of claim 23, wherein saidnucleic acid sequence encodes a polypeptide comprising the amino acidsequence depicted at positions 16-272 of SEQ ID NO:8.
 36. Thepolypeptide of claim 23, wherein said nucleic acid sequence encodes apolypeptide that comprises a polypeptide comprising an amino acidsequence that has at least 80% sequence identity to the amino acidsequence depicted at positions 21-272 of SEQ ID NO:8.
 37. Thepolypeptide of claim 23, wherein said nucleic acid sequence encodes apolypeptide that consists of an amino acid sequence that has at least80% sequence identity to the amino acid sequence depicted at positions21-272 of SEQ ID NO:8.
 38. The polypeptide of claim 23, wherein saidnucleic acid sequence encodes a polypeptide that consists of an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence depicted at positions 21-272 of SEQ ID NO:8.
 39. Thepolypeptide of claim 23, wherein said nucleic acid sequence encodes apolypeptide that consists of an amino acid sequence that has at least95% sequence identity to the amino acid sequence depicted at positions21-272 of SEQ ID NO:8.
 40. The polypeptide of claim 23, wherein saidnucleic acid sequence encodes a polypeptide that consists of the aminoacid sequence depicted at positions 21-272 of SEQ ID NO:8.
 41. Thepolypeptide of claim 23, wherein said nucleic acid sequence encodes apolypeptide comprising the amino acid sequence depicted at positions21-272 of SEQ ID NO:8.
 42. The polypeptide of claim 23, wherein saidnucleic acid sequence encodes a polypeptide comprising a fragment of theamino acid sequence depicted at positions 1-272 of SEQ ID NO:8 andwherein said fragment comprises at least 25 consecutive amino acidsthereof.
 43. A purified insulin-like growth factor binding protein-5(IGFBP-5) having the amino acid sequence of SEQ ID NO:8.
 44. Arecombinant DNA molecule comprising a nucleic acid sequence encoding apolypeptide according to claim
 1. 45. The recombinant DNA molecule ofclaim 44, wherein the nucleic acid sequence encodes a polypeptide thatcomprises a polypeptide comprising an amino acid sequence that has atleast 80% sequence identity to the amino acid sequence depicted atpositions 1-272 of SEQ ID NO:8.
 46. The recombinant DNA molecule ofclaim 44, wherein the nucleic acid sequence encodes a polypeptide thatconsists of an amino acid sequence that has at least 80% sequenceidentity to the amino acid sequence depicted at positions 1-272 of SEQID NO:8.
 47. The recombinant DNA molecule of claim 44, wherein thenucleic acid sequence encodes a polypeptide that consists of an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence depicted at positions 1-272 of SEQ ID NO:8.
 48. The recombinantDNA molecule of claim 44, wherein the nucleic acid sequence encodes apolypeptide that consists of an amino acid sequence that has at least95% sequence identity to the amino acid sequence depicted at positions1-272 of SEQ ID NO:8.
 49. The recombinant DNA molecule of claim 44,wherein the nucleic acid sequence encodes a polypeptide that consists ofthe amino acid sequence depicted at positions 1-272 of SEQ ID NO:8. 50.The recombinant DNA molecule of claim 44, wherein the nucleic acidsequence encodes a polypeptide comprising the amino acid sequencedepicted at positions 1-272 of SEQ ID NO:8.
 51. The recombinant DNAmolecule of claim 44, wherein the nucleic acid sequence encodes apolypeptide that comprises a polypeptide comprising an amino acidsequence that has at least 80% sequence identity to the amino acidsequence depicted at positions 16-272 of SEQ ID NO:8.
 52. Therecombinant DNA molecule of claim 44, wherein the nucleic acid sequenceencodes a polypeptide that consists of an amino acid sequence that hasat least 80% sequence identity to the amino acid sequence depicted atpositions 16-272 of SEQ ID NO:8.
 53. The recombinant DNA molecule ofclaim 44, wherein the nucleic acid sequence encodes a polypeptide thatconsists of an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence depicted at positions 16-272 of SEQID NO:8.
 54. The recombinant DNA molecule of claim 44, wherein thenucleic acid sequence encodes a polypeptide that consists of an aminoacid sequence that has at least 95% sequence identity to the amino acidsequence depicted at positions 16-272 of SEQ ID NO:8.
 55. Therecombinant DNA molecule of claim 44, wherein the nucleic acid sequenceencodes a polypeptide that consists of the amino acid sequence depictedat positions 16-272 of SEQ ID NO:8.
 56. The recombinant DNA molecule ofclaim 44, wherein the nucleic acid sequence encodes a polypeptidecomprising the amino acid sequence depicted at positions 16-272 of SEQID NO:8.
 57. The recombinant DNA molecule of claim 44, wherein thenucleic acid sequence encodes a polypeptide that comprises a polypeptidecomprising an amino acid sequence that has at least 80% sequenceidentity to the amino acid sequence depicted at positions 21-272 of SEQID NO:8.
 58. The recombinant DNA molecule of claim 44, wherein thenucleic acid sequence encodes a polypeptide that consists of an aminoacid sequence that has at least 80% sequence identity to the amino acidsequence depicted at positions 21-272 of SEQ ID NO:8.
 59. Therecombinant DNA molecule of claim 44, wherein the nucleic acid sequenceencodes a polypeptide that consists of an amino acid sequence that hasat least 90% sequence identity to the amino acid sequence depicted atpositions 21-272 of SEQ ID NO:8.
 60. The recombinant DNA molecule ofclaim 44, wherein the nucleic acid sequence encodes a polypeptide thatconsists of an amino acid sequence that has at least 95% sequenceidentity to the amino acid sequence depicted at positions 21-272 of SEQID NO:8.
 61. The recombinant DNA molecule of claim 44, wherein thenucleic acid sequence encodes a polypeptide that consists of the aminoacid sequence depicted at positions 21-272 of SEQ ID NO:8.
 62. Therecombinant DNA molecule of claim 44, wherein the nucleic acid sequenceencodes a polypeptide comprising the amino acid sequence depicted atpositions 21-272 of SEQ ID NO:8.
 63. The recombinant DNA molecule ofclaim 44, wherein the nucleic acid sequence encodes a polypeptidecomprising a fragment of the amino acid sequence depicted at positions1-272 of SEQ ID NO:8 and wherein said fragment comprises at least 25consecutive amino acids thereof.
 64. The recombinant DNA molecule ofclaims 44, wherein the molecule comprises the nucleic acid sequencedepicted at nucleotide positions 1-859 of SEQ ID NO:7.
 65. Therecombinant DNA molecule of claim 44, wherein the molecule comprises thenucleic acid sequence depicted at nucleotide positions 89-859 of SEQ IDNO:7.
 66. The recombinant DNA molecule of claim 44, wherein the moleculecomprises the nucleic acid sequence depicted at nucleotide positions104-859 of SEQ ID NO:7.
 67. The recombinant DNA molecule of claim 44,wherein the sequence is a human DNA sequence.
 68. The recombinant DNAmolecule of claim 44, wherein the sequence is a genomic sequence. 69.The recombinant DNA molecule of claim 44, wherein the sequence is a cDNAsequence.
 70. A recombinant DNA expression vector comprising the DNA ofclaim 44, wherein the vector is capable of effecting the expression ofthe polypeptide in a microorganism.
 71. The recombinant DNA molecule ofclaim 44, wherein the DNA molecule is contained in pBsBP6.1.
 72. Anexpression vector comprising the DNA molecule of claim 44 and aregulatory sequence for expression of the polypeptide.
 73. Theexpression vector as claimed in claim 72, wherein the vector is operablefor expression in an insect cell.
 74. The expression vector as claimedin claim 72, wherein the vector is operable for expression in a yeasthost.
 75. The expression vector as claimed in claim 72, wherein thevector is operable for expression in a bacterial host.
 76. Theexpression vector as claimed in claim 72, wherein the regulatorysequence comprises a promoter sequence selected from the groupconsisting of ADH2/GAPDH and GAPDH promoter sequences.
 77. Theexpression vector as claimed in claim 76 wherein the vector furthercomprises east a fragment of a pre-pro alpha-factor leader sequencesufficient for secretion.
 78. A host cell transformed with theexpression vector of claim
 72. 79. The host cell as claimed in claim 78,wherein the cell is selected from the group consisting of a bacterialcell, a yeast cell, a mammalian cell and an insect cell.
 80. Arecombinant microorganism or cell line containing the DNA molecule ofclaim
 44. 81. The microorganism of claim 80, wherein the microorganismis a yeast.
 82. The cell line of claim 80, wherein the cell line is aCHO cell line.
 83. A method for producing a recombinant polypeptidecomprising the steps of culturing a recombinant host transformed by theDNA molecule of claim 44 under conditions that allow the expression ofthe polypeptide; and isolating the expressed polypeptide.
 84. The methodof claim 83, wherein the host is a microorganism.
 85. The method ofclaim 83, wherein the host is a eucaryotic cell.                   #             SEQUENCE LISTING (1) GENERAL INFORMATION:   (iii) NUMBER OF SEQUENCES: 8 (2) INFORMATION FOR SEQ ID NO:1:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 14 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid          (A) DESCRIPTION: /desc  #= “oligonucleotide”    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:CCTGTAGATC TCCG               #                   #                  #     14 (2) INFORMATION FOR SEQ ID NO:2:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 18 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid          (A) DESCRIPTION: /desc  #= “oligonucleotide”    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:AATTCGGAGA TCTACAGG              #                   #                  #  18 (2) INFORMATION FOR SEQ ID NO:3:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 20 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid          (A) DESCRIPTION: /desc  #= “oligonucleotide”    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:AGATCTGAAT TCGCCAATGA             #                  #                   # 20 (2) INFORMATION FOR SEQ ID NO:4:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 22 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid          (A) DESCRIPTION: /desc  #= “oligonucleotide”    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:AGATCTAAGC TTCACCACCA CA            #                  #                 22 (2) INFORMATION FOR SEQ ID NO:5:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 19 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid          (A) DESCRIPTION: /desc  #= “oligonucleotide”    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:GCAAAGGATT CTACAAGAG             #                   #                  #  19 (2) INFORMATION FOR SEQ ID NO:6:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 19 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid          (A) DESCRIPTION: /desc  #= “oligonucleotide”    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:CAAACCTTCC CGTGGCCGC             #                   #                  #  19 (2) INFORMATION FOR SEQ ID NO:7:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 1612 base #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:CTCTCCTGCC CCACCCCGAG GTAAAGGGGG CGACTAAGAG AAGATGGTGT T#GCTCACCGC     60GGTCCTCCTG CTGCTGGCCG CCTATGCGGG GCCGGCCCAG AGCCTGGGCT C#CTTCGTGCA    120CTGCGAGCCC TGCGACGAGA AAGCCCTCTC CATGTGCCCC CCCAGCCCCC T#GGGCTGCGA    180GCTGGTCAAG GAGCCGGGCT GCGGCTGCTG CATGACCTGC GCCCTGGCCG A#GGGGCAGTC    240GTGCGGCGTC TACACCGAGC GCTGCGCCCA GGGGCTGCGC TGCCTCCCCC G#GCAGGACGA    300GGAGAAGCCG CTGCACGCCC TGCTGCACGG CCGCGGGGTT TGCCTCAACG A#AAAGAGCTA    360CCGCGAGCAA GTCAAGATCG AGAGAGACTC CCGTGAGCAC GAGGAGCCCA C#CACCTCTGA    420GATGGCCGAG GAGACCTACT CCCCCAAGAT CTTCCGGCCC AAACACACCC G#CATCTCCGA    480GCTGAAGGCT GAAGCAGTGA AGAAGGACCG CAGAAAGAAG CTGACCCAGT C#CAAGTTTGT    540CGGGGGAGCC GAGAACACTG CCCACCCCCG GATCATCTCT GCACCTGAGA T#GAGACAGGA    600GTCTGAGCAG GGCCCCTGCC GCAGACACAT GGAGGCTTCC CTGCAGGAGC T#CAAAGCCAG    660CCCACGCATG GTGCCCCGTG CTGTGTACCT GCCCAATTGT GACCGCAAAG G#ATTCTACAA    720GAGAAAGCAG TGCAAACCTT CCCGTGGCCG CAAGCGTGGC ATCTGCTGGT G#CGTGGACAA    780GTACGGGATG AAGCTGCCAG GCATGGAGTA CGTTGACGGG GACTTTCAGT G#CCACACCTT    840CGACAGCAGC AACGTTGAGT GATGCGTCCC CCCCCAACCT TTCCCTCACC C#CCTCCCACC    900CCCAGCCCCG ACTCCAGCCA GCGCCTCCCT CCACCCCAGG ACGCCACTCA T#TTCATCTCA    960TTTAAGGGAA AAATATATAT CTATCTATTT GAGGAAACTG AGGACCTCGG A#ATCTCTAGC   1020AAGGGCTCAA CTTCGAAAAT GGCAACAACA GAGATGCAAA AAGCTAAAAA G#ACACCCCCC   1080CCCTTTAAAT GGTTTTCTTT TTGAGGCAAG TTGGATGAAC AGAGAAGGGA A#GAGAGGAAG   1140AACGAGAGGA AGAGAAGGGA AGGAAGTGTT TGTGTAGAAG AGAGAGAAAG A#CGAATAGAG   1200TTAGGAAAAG GAAGACAAGC AGGTGGGCAG GAAGGACATG CACCGAGACC A#GGCAGGGGC   1260CCAACTTTCA CGTCCAGCCC TGGCCTGGGG TCGGGAGAGG TGGGCGCTAG A#AGATGCAGC   1320CCAGGATGTG GCAATCAATG ACACTATTGG GGTTTCCCAG GATGGATTGG T#CAGGGGGAG   1380AAAGGAAAAG GCAAAACACT CCAGGACCTC TCCCGGATCT GTCTCCTCCT C#TAGCCAGCA   1440GTATGGACAG CTGGACCCCT GAACTTCCTC TCCTCTTACC TGGGCAGAGT G#TTGTCTCTC   1500CCCAAATTTA TAAAAACTAA AATGCATTCC ATTCCTCTGA AAGCAAAACA A#ATTCATAAT   1560TGAGTGATAT TAAATAGAGA GGTTTTCGGA AGCAGATCTG TGAATATGAA A#T           1612 (2) INFORMATION FOR SEQ ID NO:8:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 272 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:Met Val Leu Leu Thr Ala Val Leu Leu Leu L #eu Ala Ala Tyr Ala Gly1               5    #                10   #                15Pro Ala Gln Ser Leu Gly Ser Phe Val His C #ys Glu Pro Cys Asp Glu            20       #            25       #            30Lys Ala Leu Ser Met Cys Pro Pro Ser Pro L #eu Gly Cys Glu Leu Val        35           #        40           #        45Lys Glu Pro Gly Cys Gly Cys Cys Met Thr C #ys Ala Leu Ala Glu Gly    50               #    55               #    60Gln Ser Cys Gly Val Tyr Thr Glu Arg Cys A #la Gln Gly Leu Arg Cys65                   #70                   #75                   #80Leu Pro Arg Gln Asp Glu Glu Lys Pro Leu H #is Ala Leu Leu His Gly                85   #                90   #                95Arg Gly Val Cys Leu Asn Glu Lys Ser Tyr A #rg Glu Gln Val Lys Ile            100      #            105      #            110Glu Arg Asp Ser Arg Glu His Glu Glu Pro T #hr Thr Ser Glu Met Ala        115          #        120          #        125Glu Glu Thr Tyr Ser Pro Lys Ile Phe Arg P #ro Lys His Thr Arg Ile    130              #    135              #    140Ser Glu Leu Lys Ala Glu Ala Val Lys Lys A #sp Arg Arg Lys Lys Leu145                  #150                  #155                  #160Thr Gln Ser Lys Phe Val Gly Gly Ala Glu A #sn Thr Ala His Pro Arg                165  #                170  #                175Ile Ile Ser Ala Pro Glu Met Arg Gln Glu S #er Glu Gln Gly Pro Cys            180      #            185      #            190Arg Arg His Met Glu Ala Ser Leu Gln Glu L #eu Lys Ala Ser Pro Arg        195          #        200          #        205Met Val Pro Arg Ala Val Tyr Leu Pro Asn C #ys Asp Arg Lys Gly Phe    210              #    215              #    220Tyr Lys Arg Lys Gln Cys Lys Pro Ser Arg G #ly Arg Lys Arg Gly Ile225                  #230                  #235                  #240Cys Trp Cys Val Asp Lys Tyr Gly Met Lys L #eu Pro Gly Met Glu Tyr                245  #                250  #                255Val Asp Gly Asp Phe Gln Cys His Thr Phe A #sp Ser Ser Asn Val Glu            260      #            265      #            270


86. A method of producing a recombinant polypeptide comprising: (a)providing a cell that comprises the isolated DNA molecule of claim 44that is operatively connected to control sequences that allowsexpression of the polypeptide; and (b) allowing the cells to produce thepolypeptide.
 87. An expression vector comprising the DNA molecule ofclaim 50 and a regulatory sequence for expression of the polypeptide.88. An expression vector comprising the DNA molecule of claim 56 and aregulatory sequence for expression of the polypeptide.
 89. An expressionvector comprising the DNA molecule of claim 62 and a regulatory sequencefor expression of the polypeptide.
 90. An expression vector comprisingthe DNA molecule of claim 63 and a regulatory sequence for expression ofthe polypeptide.
 91. A host cell transformed with the expression vectorof claim
 87. 92. A host cell transformed with the expression vector ofclaim
 88. 93. A host cell transformed with the expression vector ofclaim
 89. 94. A host cell transformed with the expression vector ofclaim
 90. 95. A method for producing a recombinant polypeptidecomprising the steps of culturing the host cell of claim 91 underconditions that allow the expression of the polypeptide; and isolatingthe expressed polypeptide.
 96. A method for producing a recombinantpolypeptide comprising the steps of culturing the host cell of claim 92under conditions that allow the expression of the polypeptide; andisolating the expressed polypeptide.
 97. A method for producing arecombinant polypeptide comprising the steps of culturing the host cellof claim 93 under conditions that allow the expression of thepolypeptide; and isolating the expressed polypeptide.
 98. A method forproducing a recombinant polypeptide comprising the steps of culturingthe host cell of claim 94 under conditions that allow the expression ofthe polypeptide; and isolating the expressed polypeptide.
 99. Anisolated DNA molecule encoding insulin-like growth factor bindingprotein-5 (IGFBP-5) having the sequence of SEQ ID NO:8.
 100. An antibodywhich specifically binds to a polypeptide according to claim
 1. 101. Theantibody of claim 100, wherein said antibody is a monoclonal antibody.